Methods and compositions relating to ex vivo culture and modulation of t cells

ABSTRACT

Described herein are methods for producing and utilizing an artificial antigen presenting cell (aAPC). An aAPC is engineered to express a first and second chimeric stimulatory receptor that specifically bind antigen presenting on a T cell of interest. The aAPC as described herein is designed for use in activating and/or expanding a T cell or chimeric antigen receptor (CAR) T cell. Further, this invention relates to methods of treating cancer by administering to a subject in need thereof an aAPC-activated CAR T cell.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit under 35 U.S.C. § 119(e) of U.S.Provisional Application Nos. 62/444,608, filed Jan. 10, 2017,62/485,679, filed Apr. 14, 2017, and 62/580,256, filed Nov. 1, 2017, thecontents of which are incorporated herein by reference in theirentirety.

TECHNICAL FIELD

The technology described herein relates to cancer immunotherapies.

BACKGROUND

T cell therapies targeting tumor antigens have shown unprecedentedcomplete remissions in patients with refractory and relapsing cancers inthe past few years. The clinical doses of these T cell products arecommonly achieved through the expansion of naive T cells by stimulationwith paramagnetic microspheres, which are covalently bound to antibodiescrosslinking key molecules signaling T cell growth, namely CD3 and CD28.Improved methods bearing the features required to generate and expandtumor-targeting T cells ex vivo for clinical use are needed.

SUMMARY

Provided herein are methods for producing an artificial antigenpresenting cell (aAPC) and utilizing the aAPC for the activation of a Tcell, including, but not limited to a chimeric antigen receptor (CAR) Tcell. Additionally provided herein, are methods for the treatment of aplasma cell disease or disorder, or an autoimmune disease or disorder byadministering an activated T cell or CAR T cell to a subject in needthereof

Accordingly, in one aspect, described herein is an artificial antigenpresenting cell (aAPC) comprising a first chimeric stimulatory receptor(CSR) that binds specifically with a first co-stimulatory peptide orpolypeptide region, and a second CSR that binds specifically with asecond co-stimulatory peptide or polypeptide region.

Another aspect described herein relates to an aAPC comprising a firstchimeric stimulatory receptor (CSR) that binds specifically with CD3,and a second CSR that binds specifically with CD28.

As used herein, “chimeric stimulatory receptor” refers to a polypeptidecomprising, from N-terminus to C-terminus an antibody reagent or anatural ligand specific for a T cell activating receptor (e.g. CD3 orCD28); a linker domain; and a transmembrane domain.

In some embodiments of any of the aspects, the aAPC has been engineeredto lack an expressible LDLR gene. In some embodiments of any of theaspects, the aAPC comprises a deletion in the native LDLR-encodingnucleic acid sequence. In other embodiments of any of the aspects, theaAPC comprises a deletion of the native LDLR-encoding nucleic acidsequence.

In some embodiments of any of the aspects, the aAPC is viable butnon-dividing.

In some embodiments of any of the aspects, the aAPC has beengamma-irradiated.

In some embodiments of any of the aspects, the aAPC is furtherengineered to comprise a T-cell target molecule. In some embodiments ofany of the aspects, the T-cell target molecule is CD19, BCMA, CD37,SLAMF7, EGFR, or EGFR variant III. In some embodiments of any of theaspects, the T-cell target molecule is CD19. In some embodiments of anyof the aspects, the T-cell target molecule is any molecule that can betargeted by a CAR T cell.

In some embodiments of any of the aspects, the first and second CSRs areexpressed on the cell surface of the aAPC.

In some embodiments of any of the aspects, the first and second CSRs areconstitutively expressed.

In some embodiments of any of the aspects, the first and second CSRs areencoded by a first recombinant nucleic acid sequence and a secondrecombinant nucleic acid sequence, respectively. In some embodiments ofany of the aspects, the sequence encoding the first and/or second CSR isoperatively linked to a constitutive promoter. In some embodiments ofany of the aspects, the constitutive promoter is an EF1-oc promoter.Non-limiting examples of additional constitutive promoters include theMND promoter and the PGK promoter.

In some embodiments of any of the aspects, the aAPC is a human cell.

In some embodiments of any of the aspects, aAPC is engineered from anerythromyeloid cell. In some embodiments of any of the aspects, theerthromyeloid cell is a K562 cell.

In some embodiments of any of the aspects, the first and second CSRsbind specifically with human CD3 and CD28, respectively.

In some embodiments of any of the aspects, the first CSR bindsspecifically with 4-1BBL, and the second CSR binds specifically withOX40L.

Another aspect of the technology described herein relates to a method ofexpanding and/or activating a T cell, the method comprising contactingan aAPC as described herein with a T cell.

Another aspect of the technology described herein relates to a method oftreating a cancer, a plasma cell disease or disorder, or an autoimmunedisease or disorder in a subject in need thereof, the method comprisingcontacting an aAPC as described herein with a CAR T cell, therebyactivating the CAR T cell, and administering the activated CAR T cell tothe subject.

In some embodiments, the contacting step occurs in vitro. In someembodiments, the contacting step occurs in suspension.

In some embodiments, the cancer is a leukemia, lymphoma, multiplemyeloma, or solid tumor. In some embodiments, the leukemia is ALL orCLL. In some embodiments, the lymphoma is follicular lymphoma or diffuselarge B cell lymphoma.

Another aspect of the technology described herein relates to acomposition comprising an aAPC as described herein and a T cell. In oneembodiment, the T cell is a CAR T cell.

Another aspect of the technology described herein relates to acomposition comprising activated CAR T cells described herein, andoptionally a pharmaceutically acceptable carrier, for the treatment ofcancer.

Definitions

For convenience, the meaning of some terms and phrases used in thespecification, examples, and appended claims, are provided below. Unlessstated otherwise, or implicit from context, the following terms andphrases include the meanings provided below. The definitions areprovided to aid in describing particular embodiments, and are notintended to limit the claimed invention, because the scope of theinvention is limited only by the claims. Unless otherwise defined, alltechnical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs. If there is an apparent discrepancy between the usageof a term in the art and its definition provided herein, the definitionprovided within the specification shall prevail.

Definitions of common terms in immunology and molecular biology can befound in The Merck Manual of Diagnosis and Therapy, 19th Edition,published by Merck Sharp & Dohme Corp., 2011 (ISBN 978-0-911910-19-3);Robert S . Porter et al. (eds.), The Encyclopedia of Molecular CellBiology and Molecular Medicine, published by Blackwell Science Ltd.,1999-2012 (ISBN 9783527600908); and Robert A. Meyers (ed.), MolecularBiology and Biotechnology: a Comprehensive Desk Reference, published byVCH Publishers, Inc., 1995 (ISBN 1-56081-569-8); Immunology by WernerLuttmann, published by Elsevier, 2006; Janeway's Immunobiology, KennethMurphy, Allan Mowat, Casey Weaver (eds.), Taylor & Francis Limited, 2014(ISBN 0815345305, 9780815345305); Lewin's Genes XI, published by Jones &Bartlett Publishers, 2014 (ISBN-1449659055); Michael Richard Green andJoseph Sambrook, Molecular Cloning: A Laboratory Manual, 4^(th) ed.,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA(2012) (ISBN 1936113414); Davis et al, Basic Methods in MolecularBiology, Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN044460149X); Laboratory Methods in Enzymology: DNA, Jon Lorsch (ed.)Elsevier, 2013 (ISBN 0124199542); Current Protocols in Molecular Biology(CPMB), Frederick M. Ausubel (ed.), John Wiley and Sons, 2014 (ISBN047150338X, 9780471503385), Current Protocols in Protein Science (CPPS),John E. Coligan (ed.), John Wiley and Sons, Inc., 2005; and CurrentProtocols in Immunology (CPI) (John E. Coligan, ADA M Kruisbeek, David HMargulies, Ethan M Shevach, Warren Strobe, (eds.) John Wiley and Sons,Inc., 2003 (ISBN 0471142735, 9780471 142737), the contents of which areall incorporated by reference herein in their entireties.

The terms “decrease,” “reduced,” “reduction,” and “inhibit” are all usedherein to mean a decrease by a statistically significant amount. In someembodiments, “reduce,” “reduction” or “decrease” or “inhibit” typicallymeans a decrease by at least 10% as compared to a reference level (e.g.the absence of a given treatment or agent) and can include, for example,a decrease by at least about 10%, at least about 20%, at least about25%, at least about 30%, at least about 35%, at least about 40%, atleast about 45%, at least about 50%, at least about 55%, at least about60%, at least about 65%, at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about95%, at least about 98%, at least about 99% , or more. As used herein,“reduction” or “inhibition” does not encompass a complete inhibition orreduction as compared to a reference level. “Complete inhibition” is a100% inhibition as compared to a reference level. Where applicable, adecrease can be preferably down to a level accepted as within the rangeof normal for an individual without a given disorder.

The terms “increased,” “increase,” “enhance,” and “activate” are allused herein to mean an increase by a statically significant amount. Insome embodiments, the terms “increased”, “increase”, “enhance”, or“activate” can mean an increase of at least 10% as compared to areference level, for example an increase of at least about 20%, or atleast about 30%, or at least about 40%, or at least about 50%, or atleast about 60%, or at least about 70%, or at least about 80%, or atleast about 90% or up to and including a 100% increase or any increasebetween 10-100% as compared to a reference level, or at least about a2-fold, or at least about a 3-fold, or at least about a 4-fold, or atleast about a 5-fold or at least about a 10-fold increase, or anyincrease between 2-fold and 10-fold or greater as compared to areference level. In the context of a marker or symptom, an “increase” isa statistically significant increase in such level.

As used herein, a “subject” means a human or animal. Usually the animalis a vertebrate such as a primate, rodent, domestic animal or gameanimal. Primates include chimpanzees, cynomologous monkeys, spidermonkeys, and macaques, e.g., Rhesus. Rodents include mice, rats,woodchucks, ferrets, rabbits and hamsters. Domestic and game animalsinclude cows, horses, pigs, deer, bison, buffalo, feline species, e.g.,domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g.,chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon. Insome embodiments, the subject is a mammal, e.g., a primate, e.g., ahuman. The terms, “individual,” “patient,” and “subject” are usedinterchangeably herein.

Preferably, the subject is a mammal. The mammal can be a human,non-human primate, mouse, rat, dog, cat, horse, or cow, but is notlimited to these examples. Mammals other than humans can beadvantageously used as subjects that represent animal models of e.g.,cancer. A subject can be male or female.

A subject can be one who has been previously diagnosed with oridentified as suffering from or having a condition in need of treatment(e.g., ALL or another type of cancer) or one or more complicationsrelated to such a condition, and optionally, have already undergonetreatment for the condition or the one or more complications related tothe condition. Alternatively, a subject can also be one who has not beenpreviously diagnosed as having such condition or related complications.For example, a subject can be one who exhibits one or more risk factorsfor the condition or one or more complications related to the conditionor a subject who does not exhibit risk factors.

A “subject in need” of treatment for a particular condition can be asubject having that condition, diagnosed as having that condition, or atrisk of developing that condition.

A “disease” is a state of health of an animal, for example a human,wherein the animal cannot maintain homeostasis, and wherein if thedisease is not ameliorated, then the animal's health continues todeteriorate. In contrast, a “disorder” in an animal is a state of healthin which the animal is able to maintain homeostasis, but in which theanimal's state of health is less favorable than it would be in theabsence of the disorder. Left untreated, a disorder does not necessarilycause a further decrease in the animal's state of health.

The term “cancer” and its grammatical equivalents as used herein canrefer to a hyperproliferation of cells whose unique trait—loss of normalcellular control—results in unregulated growth, lack of differentiation,local tissue invasion, and metastasis. With respect to the inventivemethods, the cancer can be any cancer recognized by a skilled person.Cancer can include, but is not limited to, any of acute lymphocyticcancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer,brain cancer, breast cancer, cancer of the anus, anal canal, rectum,cancer of the eye, cancer of the intrahepatic bile duct, cancer of thejoints, cancer of the neck, gallbladder, or pleura, cancer of the nose,nasal cavity, or middle ear, cancer of the oral cavity, cancer of thevulva, chronic lymphocytic leukemia, chronic myeloid cancer, coloncancer, esophageal cancer, cervical cancer, fibrosarcoma,gastrointestinal carcinoid tumor, Hodgkin lymphoma, hypopharynx cancer,kidney cancer, larynx cancer, liquid tumors, liver cancer, lung cancer,lymphoma, malignant mesothelioma, mastocytoma, melanoma, multiplemyeloma, nasopharynx cancer, non-Hodgkin lymphoma, ovarian cancer,pancreatic cancer, peritoneum, omentum, and mesentery cancer, pharynxcancer, prostate cancer, skin cancer, small intestine cancer, softtissue cancer, solid tumors, stomach cancer, testicular cancer, thyroidcancer, ureter cancer, and/or urinary bladder cancer. As used herein,the term “tumor” refers to an abnormal growth of cells or tissues, e.g.,of malignant type or benign type.

An “autoimmune disease or disorder” is characterized by the inability ofone's immune system to distinguish between a foreign cell and a healthycell of one's own body. This results in one's immune system mounting aresponse to target one's healthy cells. Non-limiting examples of anautoimmune disease or disorder include rheumatoid arthritis, multiplesclerosis (MS), systemic lupus erythematosus, Graves' disease(overactive thyroid), Hashimoto's thyroiditis (underactive thyroid),celiac disease, Crohn's disease and ulcerative colitis, Guillain-Barresyndrome, primary biliary sclerosis/cirrhosis, sclerosing cholangitis,autoimmune hepatitis, Raynaud's phenomenon, scleroderma, Sjogren'ssyndrome, Goodpasture's syndrome, Wegener's granulomatosis, polymyalgiarheumatica, temporal arteritis/giant cell arteritis, chronic fatiguesyndrome CFS), psoriasis, autoimmune Addison's Disease, ankylosingspondylitis, Acute disseminated encephalomyelitis, antiphospholipidantibody syndrome, aplastic anemia, idiopathic thrombocytopenic purpura,Myasthenia gravis, opsoclonus myoclonus syndrome, optic neuritis, Ord'sthyroiditis, pemphigus, pernicious anaemia, polyarthritis in dogs,Reiter's syndrome, Takayasu' s arteritis, warm autoimmune hemolyticanemia, Wegener's granulomatosis and fibromyalgia (FM).

A plasma cell is a white blood cell produces from B lymphocytes whichfunction to generate and release antibodies needed to fight infections.A “plasma cell disease or disorder” is characterized by abnormalmultiplication of a plasma cell. Abnormal plasma cells are capable of“crowding out” healthy plasma cells, which results in a decreasedcapacity to fight a foreign object, such as a virus or bacterial cell.Non-limiting examples of plasma cell disorders include amyloidosis,Waldenstrom's macroglobulinemia, osteosclerotic myeloma (POEMSsyndrome), Monoclonal gammopathy of unknown significance (MGUS), andplasma cell myeloma.

As used herein, the terms “tumor antigen” and “cancer antigen” are usedinterchangeably to refer to antigens which are differentially expressedby cancer cells and can thereby be exploited in order to target cancercells. Cancer antigens are antigens which can potentially stimulatesubstantially tumor-specific immune responses. Some of these antigensare encoded, although not necessarily expressed, by normal cells. Theseantigens can be characterized as those which are normally silent (i.e.,not expressed) in normal cells, those that are expressed only at certainstages of differentiation and those that are temporally expressed suchas embryonic and fetal antigens found on or expressed by cancer cells.Other cancer antigens are encoded by mutant cellular genes, such asoncogenes (e.g., activated ras oncogene), suppressor genes (e.g., mutantp53), and fusion proteins resulting from internal deletions orchromosomal translocations. Still other cancer antigens can be encodedby viral genes such as those carried on RNA and DNA tumor viruses. Manytumor antigens have been defined in terms of multiple solid tumors: MAGE1, 2, & 3, defined by immunity; MART-1/Melan-A, gplOO, carcinoembryonicantigen (CEA), HER2, mucins (i.e., MUC-1), prostate-specific antigen(PSA), and prostatic acid phosphatase (PAP). Tumor antigens readilytargeted by T cells, such as CAR T cells, are generally expressed on thesurface of a cancer cell.

As used herein, the term “chimeric” refers to the product of the fusionof portions of at least two or more different polynucleotide molecules.In one embodiment, the term “chimeric” refers to a gene expressionelement produced through the manipulation of known elements or otherpolynucleotide molecules

In some embodiments, “activation” can refer to the state of a T cellthat has been sufficiently stimulated to induce detectable cellularproliferation. In some embodiments activation can refer to inducedcytokine production. In other embodiments, activation can refer todetectable effector functions. At a minimum, an “activated T cells” asused herein is a proliferative T cell. In one embodiment, an activated Tcell can be assessed by its cell-surface molecule profile. Non-limitingexamples of molecules expressed the surface of an activated T cellinclude CD25, 4-IBB, and HLA-DR. Activated T cells also secretecytokines, including, but not limited to IL-2. Methods to identify thesesurface molecules and secreted cytokines are known in the art.

As used herein, the terms “specific binding,” “binds specifically,” and“specifically binds” refer to a physical interaction between twomolecules, compounds, cells and/or particles wherein the first entitybinds to the second, target entity with greater specificity and affinitythan it binds to a third entity which is a non-target. In someembodiments, specific binding can refer to an affinity of the firstentity for the second target, entity, which is at least 10 times, atleast 50 times, at least 100 times, at least 500 times, at least 1000times or more greater than the affinity for the third non-target entityunder the same conditions. A reagent specific for a given target is onethat exhibits specific binding for that target under the conditions ofthe assay being utilized. A non-limiting example includes an antibody ora ligand, which recognizes and binds with a cognate binding partner (forexample, a stimulatory and/or costimulatory molecule present on a Tcell) protein.

A “stimulatory ligand,” as used herein, refers to a ligand that whenpresent on an antigen presenting cell (e.g., an aAPC, a dendritic cell,a B-cell, and the like) can specifically bind with a cognate bindingpartner (referred to herein as a “stimulatory molecule” or“co-stimulatory molecule”) on a T cell, thereby mediating a primaryresponse by the T cell, including, but not limited to, proliferation,activation, initiation of an immune response, and the like. Stimulatoryligands are well-known in the art and encompass, inter alia, an MHCClass I molecule loaded with a peptide, an anti-CD3 antibody, asuperagonist anti-CD28 antibody, and a superagonist anti-CD2 antibody.

A “stimulatory molecule,” as the term is used herein, means a moleculeon a T cell that specifically binds with a cognate stimulatory ligandpresent on an antigen presenting cell (including an aAPC as describedherein).

“Co-stimulatory ligand,” as the term is used herein, includes a moleculeon an antigen presenting cell (e.g., an aAPC, dendritic cell, B cell,and the like) that specifically binds a cognate co-stimulatory moleculeon a T cell, thereby providing a signal which, in addition to theprimary signal provided by, for instance, binding of a TCR/CD3 complexwith an MHC molecule loaded with peptide, mediates a T cell response,including, but not limited to, proliferation, activation,differentiation, and the like. A co-stimulatory ligand can include, butis not limited to, 4-1BBL, OX40L, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1,PD-L2, inducible COStimulatory ligand (ICOS-L), intercellular adhesionmolecule (ICAM), CD3OL, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM,lymphotoxin beta receptor, 3/TR6, ILT3, ILT4, HVEM, an agonist orantibody that binds Toll-like receptor and a ligand that specificallybinds with B7-H3. A co-stimulatory ligand also can include, but is notlimited to, an antibody that specifically binds with a co-stimulatorymolecule present on a T cell, such as, but not limited to, CD27, CD28,4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associatedantigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand thatspecifically binds with CD83.

A “co-stimulatory molecule” refers to the cognate binding partner on a Tcell that specifically binds with a co-stimulatory ligand, therebymediating a co- stimulatory response by the T cell, such as, but notlimited to, proliferation. Co-stimulatory molecules include, but are notlimited to an MHC class I molecule, BTLA, a Toll-like receptor, CD27,CD28, 4-IBB, OX40, CD30, CD40, PD-1, ICOS, lymphocytefunction-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3,and CD83.

In one embodiment, the term “engineered” and its grammatical equivalentsas used herein can refer to one or more human-designed alterations of anucleic acid, e.g., the nucleic acid within an organism's genome. Inanother embodiment, engineered can refer to alterations, additions,and/or deletion of genes. An “engineered cell” can refer to a cell withan added, deleted and/or altered gene. The term “cell” or “engineeredcell” and their grammatical equivalents as used herein can refer to acell of human or non-human animal origin.

“Chimeric antigen receptor” or “CAR” or “CARs” as used herein refers toengineered receptors, which graft an antigen specificity onto cells (forexample T cells such as naïve T cells, central memory T cells, effectormemory T cells or combination thereof). CARs are also known asartificial T-cell receptors, chimeric T-cell receptors or chimericimmunoreceptors. In one embodiment, the CARs useful in the technologydescribed herein comprise at least two antigen-specific targetingregions, an extracellular domain, a transmembrane domain, and anintracellular signaling domain. In such embodiments, the two or moreantigen- specific targeting regions target at least two differentantigens and may be arranged in tandem and separated by linkersequences. In another embodiment, the CAR is a bispecific CAR. Abispecific CAR is specific to two different antigens.

As used herein, a “CAR T cell” refers to a T cell which expresses a CAR.When expressed in a T cell, CARS have the ability to redirect T-cellspecificity and reactivity toward a selected target in anon-MHC-restricted manner, exploiting the antigen-binding properties ofmonoclonal antibodies. The non-MHC-restricted antigen recognition givesT-cells expressing CARS the ability to recognize an antigen independentof antigen processing, thus bypassing a major mechanism of tumor escape.

In one embodiment, the CAR's extracellular binding domain is composed ofa single chain Fv fragment (scFv) derived from fusing the variable heavyand light regions of a monoclonal antibody. Alternatively, scFvs may beused that are derived from Fab's (instead of from an antibody, e.g.,obtained from Fab libraries), in various embodiments, this scFv is fusedto a transmembrane domain and then to an intracellular signaling domain.“First-generation” CARs include those that solely provide CD3zeta (CD3ζ)signals upon antigen binding, “Second-generation” CARs include thosethat provide both costimulation (e.g., CD28 or CD 137) and activation(CD3ζ). “Third-generation” CARs include those that provide multiplecostimulatory (e.g., CD28 and CD 137) domains and activation domains(e.g., CD3ζ). In various embodiments, the CAR is selected to have highaffinity or avidity for the antigen. A more detailed description of CARsand CAR T cells can be found in Maus et al. Blood 2014 123:2624-35;Reardon et al. Neuro-Oncology 2014 16:1441-1458; Hoyos et al.Haematologica 2012 97:1622; Byrd et al. J Clin Oncol 2014 32:3039-47;Maher et al. Cancer Res 2009 69:4559-4562; and Tamada et al. Clin CancerRes 2012 18:6436-6445; each of which is incorporated by reference hereinin its entirety.

An “antigen presenting cell” (APC) is a cell that is capable ofactivating T cells by presenting an antigen and necessary co-stimulatoryligand to promote T cell activation, and includes, but is not limitedto, monocytes/macrophages, B cells and dendritic cells. As used herein,“artificial” refers to something made or produced, rather than occurringnaturally. As used herein, “artificial antigen presenting cell” (aAPC)refers to a cell which has been engineered to copy or mimic at least onefunction of a naturally occurring APC. In one embodiment, the aAPC canat least stimulate proliferation of a T cell or CAR T cell. In anotherembodiment, the aAPC can stimulate proliferation and/or activation of aT cell or CAR T cell.

As used herein, the term “operably linked” refers to a firstpolynucleotide molecule, such as a promoter, connected with a secondtranscribable polynucleotide molecule, such as a gene of interest, wherethe polynucleotide molecules are so arranged that the firstpolynucleotide molecule affects the function of the secondpolynucleotide molecule. The two polynucleotide molecules may or may notbe part of a single contiguous polynucleotide molecule and may or maynot be adjacent. For example, a promoter is operably linked to a gene ofinterest if the promoter regulates or mediates transcription of the geneof interest in a cell. In one embodiment, the promoter is the EFl-ocpromoter. In an alternative embodiment, the promoter is the MND promoteror the PGK promoter.

In the various embodiments described herein, it is further contemplatedthat variants (naturally occurring or otherwise), alleles, homologs,conservatively modified variants, and/or conservative substitutionvariants of any of the particular polypeptides described areencompassed. As to amino acid sequences, one of ordinary skill willrecognize that individual substitutions, deletions or additions to anucleic acid, peptide, polypeptide, or protein sequence which alters asingle amino acid or a small percentage of amino acids in the encodedsequence is a “conservatively modified variant” where the alterationresults in the substitution of an amino acid with a chemically similaramino acid and retains the desired activity of the polypeptide. Suchconservatively modified variants are in addition to and do not excludepolymorphic variants, interspecies homologs, and alleles consistent withthe disclosure.

A given amino acid can be replaced by a residue having similarphysiochemical characteristics, e.g., substituting one aliphatic residuefor another (such as Ile, Val, Leu, or Ala for one another), orsubstitution of one polar residue for another (such as between Lys andArg; Glu and Asp; or Gin and Asn). Other such conservativesubstitutions, e.g., substitutions of entire regions having similarhydrophobicity characteristics, are well known. Polypeptides comprisingconservative amino acid substitutions can be tested in any one of theassays described herein to confirm that a desired activity, e.g. CSRactivity and specificity of a native or reference polypeptide isretained.

Amino acids can be grouped according to similarities in the propertiesof their side chains (in A. L. Lehninger, in Biochemistry, second ed.,pp. 73-75, Worth Publishers, New York (1975)): (1) non-polar: Ala (A),Val (V), Leu (L), Tie (I), Pro (P), Phe (F), Trp (W), Met (M); (2)uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N),Gin (Q); (3) acidic: Asp (D), Glu (E); (4) basic: Lys (K), Arg (R), His(H). Alternatively, naturally occurring residues can be divided intogroups based on common side-chain properties: (1) hydrophobic:Norleucine, Met, Ala, Val, Leu, lie; (2) neutral hydrophilic: Cys, Ser,Thr, Asn, Gin; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5)residues that influence chain orientation: Gly, Pro; (6) aromatic: Trp,Tyr, Phe. Non-conservative substitutions will entail exchanging a memberof one of these classes for another class. Particular conservativesubstitutions include, for example; Ala into Gly or into Ser; Arg intoLys; Asn into Gin or into His; Asp into Glu; Cys into Ser; Gin into Asn;Glu into Asp; Gly into Ala or into Pro; His into Asn or into Gin; Ileinto Leu or into Val; Leu into He or into Val; Lys into Arg, into Gin orinto Glu; Met into Leu, into Tyr or into He; Phe into Met, into Leu orinto Tyr; Ser into Thr; Thr into Ser; Trp into Tyr; Tyr into Trp; and/orPhe into Val, into He or into Leu.

In some embodiments, the polypeptide described herein (or a nucleic acidencoding such a polypeptide) can be a functional fragment of one of theamino acid sequences described herein. As used herein, a “functionalfragment” is a fragment or segment of a peptide which retains at least50% of the wildtype reference polypeptide's activity according to anassay known in the art or described below herein. A functional fragmentcan comprise conservative substitutions of the sequences disclosedherein.

In some embodiments, the polypeptide described herein can be a variantof a sequence described herein. In some embodiments, the variant is aconservatively modified variant. Conservative substitution variants canbe obtained by mutations of native nucleotide sequences, for example. A“variant,” as referred to herein, is a polypeptide substantiallyhomologous to a native or reference polypeptide, but which has an aminoacid sequence different from that of the native or reference polypeptidebecause of one or a plurality of deletions, insertions or substitutions.Variant polypeptide-encoding DNA sequences encompass sequences thatcomprise one or more additions, deletions, or substitutions ofnucleotides when compared to a native or reference DNA sequence, butthat encode a variant protein or fragment thereof that retains activityof the non-variant polypeptide. A wide variety of PCR-basedsite-specific mutagenesis approaches are known in the art and can beapplied by the ordinarily skilled artisan.

A variant amino acid or DNA sequence can be at least 90%, at least 91%,at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or more, identical to a native orreference sequence. The degree of homology (percent identity) between anative and a mutant sequence can be determined, for example, bycomparing the two sequences using freely available computer programscommonly employed for this purpose on the world wide web (e.g., BLASTpor BLASTn with default settings).

Alterations of the native amino acid sequence can be accomplished by anyof a number of techniques known to one of skill in the art. Mutationscan be introduced, for example, at particular loci by synthesizingoligonucleotides containing a mutant sequence, flanked by restrictionsites permitting ligation to fragments of the native sequence. Followingligation, the resulting reconstructed sequence encodes an analog havingthe desired amino acid insertion, substitution, or deletion.Alternatively, oligonucleotide-directed site-specific mutagenesisprocedures can be employed to provide an altered nucleotide sequencehaving particular codons altered according to the substitution,deletion, or insertion required. Techniques for making such alterationsare very well established and include, for example, those disclosed byWalder et al. (Gene 42:133, 1986); Bauer et al. (Gene 37:73, 1985);Craik (BioTechniques, January 1985, 12-19); Smith et al. (GeneticEngineering: Principles and Methods, Plenum Press, 1981); and U.S. Pat.Nos. 4,518,584 and 4,737,462, which are herein incorporated by referencein their entireties. Any cysteine residue not involved in maintainingthe proper conformation of the polypeptide also can be substituted,generally with serine, to improve the oxidative stability of themolecule and prevent aberrant crosslinking. Conversely, cysteine bond(s)can be added to the polypeptide to improve its stability or facilitateoligomerization.

As used herein, the term “DNA” is defined as deoxyribonucleic acid. Theterm “polynucleotide” is used herein interchangeably with “nucleic acid”to indicate a polymer of nucleosides. Typically a polynucleotide iscomposed of nucleosides that are naturally found in DNA or RNA (e.g.,adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine,deoxythymidine, deoxyguanosine, and deoxycytidine) joined byphosphodiester bonds. However the term encompasses molecules comprisingnucleosides or nucleoside analogs containing chemically or biologicallymodified bases, modified backbones, etc., whether or not found innaturally occurring nucleic acids, and such molecules may be preferredfor certain applications. Where this application refers to apolynucleotide it is understood that both DNA, RNA, and in each caseboth single- and double-stranded forms (and complements of eachsingle-stranded molecule) are provided. “Polynucleotide sequence” asused herein can refer to the polynucleotide material itself and/or tothe sequence information (i.e. the succession of letters used asabbreviations for bases) that biochemically characterizes a specificnucleic acid. A polynucleotide sequence presented herein is presented ina 5′ to 3′ direction unless otherwise indicated.

The term “ polypeptide” as used herein refers to a polymer of aminoacids. The terms “protein” and “polypeptide” are used interchangeablyherein. A peptide is a relatively short polypeptide, typically betweenabout 2 and 60 amino acids in length. Polypeptides used herein typicallycontain amino acids such as the 20 L-amino acids that are most commonlyfound in proteins. However, other amino acids and/or amino acid analogsknown in the art can be used. One or more of the amino acids in apolypeptide may be modified, for example, by the addition of a chemicalentity such as a carbohydrate group, a phosphate group, a fatty acidgroup, a linker for conjugation, functionalization, etc. A polypeptidethat has a nonpolypeptide moiety covalently or noncovalently associatedtherewith is still considered a “polypeptide.” Exemplary modificationsinclude glycosylation and palmitoylation. Polypeptides can be purifiedfrom natural sources, produced using recombinant DNA technology orsynthesized through chemical means such as conventional solid phasepeptide synthesis, etc. The term “polypeptide sequence” or “amino acidsequence” as used herein can refer to the polypeptide material itselfand/or to the sequence information (i.e., the succession of letters orthree letter codes used as abbreviations for amino acid names) thatbiochemically characterizes a polypeptide. A polypeptide sequencepresented herein is presented in an N-terminal to C-terminal directionunless otherwise indicated.

In some embodiments, a nucleic acid encoding a polypeptide as describedherein (e.g. a CAR polypeptide) is comprised by a vector. In some of theaspects described herein, a nucleic acid sequence encoding a givenpolypeptide as described herein, or any module thereof, is operablylinked to a vector. The term “vector,” as used herein, refers to anucleic acid construct designed for delivery to a host cell or fortransfer between different host cells. As used herein, a vector can beviral or non-viral. The term “vector” encompasses any genetic elementthat is capable of replication when associated with the proper controlelements and that can transfer gene sequences to cells. A vector caninclude, but is not limited to, a cloning vector, an expression vector,a plasmid, phage, transposon, cosmid, artificial chromosome, virus,virion, etc.

As used herein, the term “expression vector” refers to a vector thatdirects expression of an RNA or polypeptide from sequences linked totranscriptional regulatory sequences on the vector. The sequencesexpressed will often, but not necessarily, be heterologous to the cell.An expression vector may comprise additional elements, for example, theexpression vector may have two replication systems, thus allowing it tobe maintained in two organisms, for example in human cells forexpression and in a prokaryotic host for cloning and amplification. Theterm “expression” refers to the cellular processes involved in producingRNA and proteins and as appropriate, secreting proteins, including whereapplicable, but not limited to, for example, transcription, transcriptprocessing, translation and protein folding, modification andprocessing. “Expression products” include RNA transcribed from a gene,and polypeptides obtained by translation of mRNA transcribed from agene. The term “gene” means the nucleic acid sequence which istranscribed (DNA) to RNA in vitro or in vivo when operably linked toappropriate regulatory sequences. The gene may or may not includeregions preceding and following the coding region, e.g. 5′ untranslated(5′UTR) or “leader” sequences and 3′ UTR or “trailer” sequences, as wellas intervening sequences (introns) between individual coding segments(exons).

As used herein, the term “viral vector” refers to a nucleic acid vectorconstruct that includes at least one element of viral origin and has thecapacity to be packaged into a viral vector particle. The viral vectorcan contain the nucleic acid encoding a polypeptide as described hereinin place of non-essential viral genes. The vector and/or particle may beutilized for the purpose of transferring nucleic acids into cells eitherin vitro or in vivo. Numerous forms of viral vectors are known in theart.

By “recombinant vector” is meant a vector that includes a heterologousnucleic acid sequence, or “transgene” that is capable of expression invivo. It should be understood that the vectors described herein can, insome embodiments, be combined with other suitable compositions andtherapies. In some embodiments, the vector is episomal. The use of asuitable episomal vector provides a means of maintaining the nucleotideof interest in the subject in high copy number extra-chromosomal DNAthereby eliminating potential effects of chromosomal integration.

As used herein, the terms “treat,” “treatment,” “treating,” and“amelioration” refer to therapeutic treatments, wherein the object is toreverse, alleviate, ameliorate, inhibit, slow down or stop theprogression or severity of a condition associated with a disease ordisorder, e.g. ALL or other cancer. The term “treating” includesreducing or alleviating at least one adverse effect or symptom of acondition, disease or disorder. Treatment is generally “effective” ifone or more symptoms or clinical markers are reduced. Alternatively,treatment is “effective” if the progression of a disease is reduced orhalted. That is, “treatment” includes not just the improvement ofsymptoms or markers, but also a cessation of, or at least slowing of,progress or worsening of symptoms compared to what would be expected inthe absence of treatment. Beneficial or desired clinical resultsinclude, but are not limited to, alleviation of one or more symptom(s),diminishment of extent of disease, stabilized (i.e., not worsening)state of disease, delay or slowing of disease progression, ameliorationor palliation of the disease state, remission (whether partial ortotal), and/or decreased mortality, whether detectable or undetectable.The term “treatment” of a disease also includes providing relief fromthe symptoms or side-effects of the disease (including palliativetreatment).

As used herein, the term “pharmaceutical composition” refers to theactive agent in combination with a pharmaceutically acceptable carriere.g. a carrier commonly used in the pharmaceutical industry. The phrase“pharmaceutically acceptable” is employed herein to refer to thosecompounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio. In some embodimentsof any of the aspects, a pharmaceutically acceptable carrier can be acarrier other than water. In some embodiments of any of the aspects, apharmaceutically acceptable carrier can be a cream, emulsion, gel,liposome, nanoparticle, and/or ointment. In some embodiments of any ofthe aspects, a pharmaceutically acceptable carrier can be an artificialor engineered carrier, e.g., a carrier in which the active ingredientwould not be found to occur in nature.

As used herein, the term “administering” refers to the placement of atherapeutic or pharmaceutical composition as disclosed herein into asubject by a method or route which results in at least partial deliveryof the agent at a desired site. Pharmaceutical compositions comprisingagents as disclosed herein can be administered by any appropriate routewhich results in an effective treatment in the subject.

The term “statistically significant” or “significantly” refers tostatistical significance and generally means a two standard deviation(2SD) or greater difference.

Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.” The term “about” when used in connection with percentages canmean +1%.

As used herein, the term “comprising” means that other elements can alsobe present in addition to the defined elements presented. The use of“comprising” indicates inclusion rather than limitation.

The term “consisting of” refers to compositions, methods, and respectivecomponents thereof as described herein, which are exclusive of anyelement not recited in that description of the embodiment.

As used herein the term “consisting essentially of” refers to thoseelements required for a given embodiment. The term permits the presenceof additional elements that do not materially affect the basic and novelor functional characteristic(s) of that embodiment of the invention.

The singular terms “a,” “an,” and “the” include plural referents unlesscontext clearly indicates otherwise. Similarly, the word “or” isintended to include “and” unless the context clearly indicatesotherwise. Although methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of thisdisclosure, suitable methods and materials are described below. Theabbreviation, “e.g.” is derived from the Latin exempli gratia, and isused herein to indicate a non-limiting example. Thus, the abbreviation“e.g.” is synonymous with the term “for example.”

Other terms are defined within the description of the various aspectsand embodiments of the technology of the following.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a diagram of the interface between a T cell and an aAPC.Both CD3 and CD28 molecules on the T cell surface are crosslinked bytheir respective chimeric stimulatory receptor expressed on the aAPCmembrane inducing T cell activation and proliferation. Unlike anti-CD3and anti-CD28-coated microspheres, genetically encoded anti-CD3 andanti-CD28 chimeric stimulatory receptors can be propagated without theneed for soluble GMP-grade antibodies, which are expensive and notwidely available.

FIG. 2 depicts a graph of population doubling of purified T cells from anormal donor and a chronic lymphocytic leukemia (CLL) patient set inculture at day zero with either CD3/CD28 microspheres (beads) or withartificial APC (aAPC) loaded with anti-CD3 and anti-CD28 monoclonalantibodies. Population doubling of T cells was calculated for days 3, 5,7, and 9 of culture.

FIG. 3 depicts a graph of proliferation of polyclonal CD8+ and CD4+ Tcells stimulated with either CD3/CD28 microspheres (beads) or withartificial APC (aAPC) loaded with anti-CD3 and anti-CD28 monoclonalantibodies as measured by [³H] thymidine incorporation between days 3and 4 of culture in the absence of cytokines. At 72 h, the cells werepulsed with [³H] thymidine and incubated for an additional 18 h beforeharvesting. Counts per minute values are shown as mean+SEM fromtriplicate cultures.

FIG. 4 depicts FACS results demonstrating that aAPCs express both OKT3and 9.3 CSRs.

FIG. 5 is a graph of T cell activation demonstrating that aAPCs induceT-cell activation (NFAT).

FIG. 6 is a graph of T cell growth demonstrating T-cell expansion usingaAPCs.

FIG. 7 is a graph of T cell growth demonstrating T-cell expansion usingaAPCs v. anti-CD3 and anti-CD28 antibody-conjugated beads (bCD3/CD28).

FIG. 8 is a graph of T-cell size analysis during culture with aAPCs v.bCD3/CD28 (3 normal donors).

FIGS. 9 and 10 depict single-cell FACS sorting, expansion and validationof productive clones.

FIG. 11 depicts LDLR KO aAPC (with OKT3 and 9.3 CSRs)-T7E1 results. 5conditions, in triplicate, are shown.

FIG. 12 depicts LDLR KO aAPC flow results.

FIG. 13A is a graph showing the results of a flow cytometry experimentvalidating the expression of CD3 CSR and CD28 CSR on LDLR KO aAPCs, asassessed by mCherry and GFP staining.

FIG. 13B is a plot showing the results of a flow cytometry experimentvalidating that LDLR is not expressed on LDLR KO aAPCs, as assessed byan anti-hLDLR-APC antibody. Unmanipulated wild-type K562 cells were usedas a positive control for LDLR expression.

FIGS. 14A and 14B demonstrate a reduction in transduction efficiency ofLDLR KO K562 cells.

FIGS. 15A and 15B are graphs showing that significantly highertransduction efficiency is achieved using aAPCs compared to bCD3/CD28.Purified T cells from four normal donors were stimulated with eitherLDLR KO aAPCs (1:1) or bCD3/CD28 (3:1) and analyzed by FACS at day 5post-stimulation (DAPI, CD3-APC, GFP, mCherry). CAR was detected bymCherry. *** indicates a p-value=0.0008.

FIG. 16 is a series of graphs showing that CAR T cells expanded withaAPCs have similar cytokine profiles to CAR T cells expanded withbCD3/CD28, except for IL-10, IL-4, and TNF-a. CAR T cells (mutl+3) froma normal donor were stimulated with K562-BCMA for LUMINEX® assay (24hours).

FIG. 17 is a graph showing that CAR T cells expanded with aAPCs havesimilar tumor killing capacity in vitro as compared to CAR T cellsexpanded with bCD3/CD28. CAR T cells from three normal donors werestimulated with U266-CBG luc (target: U266 multiple myeloma cells) andanalyzed for specific lysis in a killing assay after 16 hours.

FIG. 18 is a graph showing that CAR T cells expanded with aAPCs havesimilar tumor killing capacity in vivo as compared to CAR T cellsexpanded with bCD3/CD28. Mice (n=5) were implanted with 106 U266multiple myeloma cells at day -7, administered with CAR T cells expandedwith aAPCs or bCD3/CD28 at day 0, and assessed by in vivo imaging ofproduced proton flux at various time points over the course of 28 dayspost-CAR T cell inoculation. UTD: untreated.

DETAILED DESCRIPTION

Embodiments of the technology described herein relate to the discoverythat an artificial antigen presenting cell (aAPC) can activate T cellsmore efficiently both in terms of time and cost, than commonly usedmethods. aAPCs as described herein are engineered to express chimericstimulatory receptors (CSR). In some embodiments, a further improvementis achieved by knock down or inactivation of LDLR expression in theaAPC. The following provides description of the methods and variousconsiderations necessary to practice the technology.

Accordingly, one aspect of the invention described herein relates to anaAPC engineered to comprise a first CSR that binds specifically withCD3; and a second CSR that binds specifically with CD28. As used herein,“chimeric stimulatory receptor” and “CSR” refers to a polypeptidecomprising: i) an antibody reagent or natural ligand specific for a Tcell co-stimulatory receptor (e.g., CD3, CD28, OX40, or 4-1BB, amongothers), or a T cell receptor (TCR); ii) a linker domain; and iii) atransmembrane domain.

An antigen presenting cell or APC is a cell which displays an antigencomplexed with major histocompatibility complexes (MHC) on its surface.A T cell will recognize these complexes via a TCR. The role of an APC isto continuously process antigens and present the antigen to a T cell.Non-limiting examples of common, naturally occuring APCs includemacrophages, B cells, and dendritic cells.

APCs are essential for the immune system to mount an effective adaptiveimmune response; the function of both cytotoxic and helper T cellsrequire APCs. Antigen presentation determines the specificity ofadaptive immunity and contributes to the immune response againstintracellular and extracellular pathogens. An APC additionally plays animportant role in priming the immune system to recognize a transformedcell, e.g., a malignant cell or tumor.

A CSR refers to a polypeptide comprising a fusion of an antibody reagentor a natural ligand that binds specifically to a TCR or co-stimulatorymolecule, a linker domain, and a transmembrane domain. In oneembodiment, the antibody reagent or natural ligand that bindsspecifically to a TCR or co-stimulatory molecule, the linker domain, andthe transmembrane domain are arranged from N-terminus to C-terminus ofthe polypeptide. In another embodiment, the antibody reagent or naturalligand that binds specifically to a TCR or co-stimulatory molecule, thelinker domain, and the transmembrane domain are arranged from C-terminusto N-terminus of the polypeptide. A CSR can be designed or engineered bya number of methods known in the art, as discussed below.

In one embodiment, the first CSR and second CSR are expressed on thesurface of the aAPC, such that the transmembrane domain spans themembrane of the aAPC, and the antibody reagent or natural ligand arepresent extracellularly, (i.e., extending outward, away from the cellmembrane) (FIG. 1 ). In one embodiment, the first CSR and second CSR areexpressed in tandem on a single receptor (e.g., a single receptor thatcomprises two different scFVs). In one embodiment, the first CSR andsecond CSR are expressed in a single construct, or from a single mRNA,e.g., use of an internal ribosomal entry site (IRES) to permittranslation of both polypeptides.

In one embodiment, the first CSR and second CSR are constitutivelyexpressed. As used herein, “constitutively expressed” refers toexpression of a gene continuously by a cell. In one embodiment, thefirst CSR and second CSR are facultatively expressed, wherein the geneis only transcribed when required, or when induced by the presence of anexogenous agent or condition.

In one embodiment, the first CSR and second CSR are encoded by a firstand second recombinant nucleic acid sequences, respectively. As usedherein, “recombinant nucleic acid sequence,” also referred to as“chimeric DNA” refers to human manipulated DNA molecules that are formedusing, for example, molecular cloning, to create nucleic acid sequencesnot found in natively in a genome. The gene product of recombinant DNAis referred to as recombinant protein. The DNA used to generaterecombinant DNA can originate from various species. In one embodiment,the first CSR and second CSR are derived from human DNA. In anotherembodiment, the first CSR and second CSR are derived from mammalian DNA.In another embodiment, the first CSR and second CSR are derived fromnon-mammalian DNA. In another embodiment, the first CSR and second CSRare derived from different species.

In one embodiments, recombinant DNA encoding a CSR is comprised in avector for delivery into an aAPC. Non-limiting examples of vectors usedfor DNA delivery into a cell include viral vectors, plasmid vectors, andcosmid vectors.

As used herein, the term “antibody reagent” refers to a polypeptide thatincludes at least one immunoglobulin variable domain or immunoglobulinvariable domain sequence and which specifically binds a given antigen.An antibody reagent can comprise an antibody or a polypeptide comprisingan antigen-binding domain of an antibody. In some embodiments of any ofthe aspects, an antibody reagent can comprise a monoclonal antibody or apolypeptide comprising an antigen-binding domain of a monoclonalantibody. For example, an antibody can include a heavy (H) chainvariable region (abbreviated herein as VH), and a light (L) chainvariable region (abbreviated herein as VL). In another example, anantibody includes two heavy (H) chain variable regions and two light (L)chain variable regions. The term “antibody reagent” encompassesantigen-binding fragments of antibodies (e.g., single chain antibodies,Fab and sFab fragments, F(ab')2, Fd fragments, Fv fragments, scFv, CDRs,and domain antibody (dAb) fragments (see, e.g. de Wildt et al., Eur J.Immunol. 1996; 26(3):629-39; which is incorporated by reference hereinin its entirety)) as well as complete antibodies. An antibody can havethe structural features of IgA, IgG, IgE, IgD, or IgM (as well assubtypes and combinations thereof). Antibodies can be from any source,including mouse, rabbit, pig, rat, and primate (human and non-humanprimate) and primatized antibodies. Antibodies also include midibodies,humanized antibodies, chimeric antibodies, and the like.

The VH and VL regions can be further subdivided into regions ofhypervariability, termed “complementarity determining regions” (“CDR”),interspersed with regions that are more conserved, termed “frameworkregions” (“FR”). The extent of the framework region and CDRs has beenprecisely defined (see, Kabat, E. A., et al. (1991) Sequences ofProteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242, and Chothia, C.et al. (1987) J. Mol. Biol. 196:901-917; which are incorporated byreference herein in their entireties). Each VH and VL is typicallycomposed of three CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4.

Antibody binding domains and ways to select and clone them are wellknown to those of ordinary skill in the art. In another embodiment, theantibody reagent is an anti-OKT3 antibody reagent and has the sequenceselected from SEQ ID NO: 27 or 31. In one embodiment, the anti-OTK3antibody reagent corresponds to the sequence of SEQ ID NO: 27 or 31; orcomprises the sequence of SEQ ID NO: 27 or 31; or comprises a sequencewith at least 80%, at least 85%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, at least 99% or greater sequence identity to thesequence of SEQ ID NO: 27 or 31.

In another embodiment, the antibody reagent is an anti-CD28 antibodyreagent and has the sequence of SEQ ID NO: 35. In one embodiment, theanti-CD28 antibody corresponds to the sequence of SEQ ID NO: 35; orcomprises the sequence of SEQ ID NO: 35; or comprises a sequence with atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99% or greater sequence identity to the sequence ofSEQ ID NO: 35.

In one embodiment, the first CSR or second CSR is fused or crosslinkedto a co-stimulatory molecule or co-stimulatory domain. As used herein,the term “co-stimulatory domain” refers to an intracellular signalingdomain of a co-stimulatory molecule. Co-stimulatory molecules are cellsurface molecules other than antigen receptors or Fc receptors thatprovide a second signal required for efficient activation and functionof T lymphocytes upon binding to antigen. Illustrative examples of suchco-stimulatory molecules include CARD1 1, CD2, CD7, CD27, CD28, CD30,CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1),CD152 (CTLA4), CD223 (LAG3), CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1),CD278 (ICOS), DAP10, LAT, NKD2C SLP76, TRIM, and ZAP70. In oneembodiment, the intracellular domain is the intracellular domain of4-1BB.

As used herein “natural ligand” refers to a naturally occurring ligandthat binds a receptor in nature. Binding of a natural ligand to areceptor will generally transduce a signal, positive or negative, aloneor in conjunction with other receptors or co- stimulatory orco-inhibiting molecules. A ligand is a molecule which specifically bindsto a portion of a protein and/or receptor as that term is definedherein. A ligand can be found on the surface of a cell or organelle, orwithin the cytoplasmic space. Ligand-protein/receptor binding can resultin the alteration of the protein and/or receptor, or activate aphysiological response, for example, the activation of a signalingpathway. In one embodiment, a CSR described herein comprises a CD28natural ligand. The CD28 natural ligand can be full length, or afragment thereof. The CD28 natural ligand can be truncated to excludeits intracellular domain. Exemplary CD28 natural ligands are describedbelow.

As used herein “linker domain” refers to an oligo- or polypeptide regionfrom about 2 to about 100 amino acids in length, which links togetherany of the domains/regions of the CSR as described herein. In someembodiment, linkers can include or be composed of flexible residues suchas glycine and serine so that the adjacent protein domains are free tomove relative to one another. Longer linkers may be used when it isdesirable to ensure that two adjacent domains do not stericallyinterfere with one another. Linkers may be cleavable or non-cleavable.Examples of cleavable linkers include 2A linkers (for example T2A),2A-like linkers or functional equivalents thereof and combinationsthereof. Non-limiting examples of linkers include linkers derived fromThosea asigna.

As used herein, a “transmembrane domain” (TM) refers to the region ofthe CSR which is inserted into or crosses the plasma membrane, e.g., ofthe aAPC. The transmembrane domain of a CSR as described herein can be atransmembrane region or fragment thereof of a transmembrane protein (forexample Type I transmembrane proteins), an artificial hydrophobicsequence, or a combination thereof. Other transmembrane domains will beapparent to those of skill in the art and can be used in connection withalternate embodiments of the invention. A selected transmembrane domainwould preferably not interfere with the intended function of the CSR. Asused in the context of transmembrane regions, “fragment thereof refersto a portion of a transmembrane region that is sufficient to anchor orattach a protein to a cell surface. In some embodiments, thetransmembrane domain of a CSR described herein comprises a transmembranedomain or fragment thereof selected from the transmembrane domain of analpha, beta, or zeta chain of a T-cell receptor, CD28, CD3 epsilon,CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86,CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD1 la, CD18), ICOS(CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80(KLRF1), CD160, CD19, IL2R beta, IL2R gamma, IL7R a, ITGA!, VLA!, CD49a,ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD! Id, ITGAE, CD103,ITGAL, CD! la, LFA-1, ITGAM, CD! lb, ITGAX, CD! lc, ITGB1, CD29, ITGB2,CD18, LFA-1, ITGB7, TNFR2, DNAM1(CD226), SLAMF4 (CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100(SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME(SLAMF8), SELPLG (CD 162), LTBR, PAG/Cbp, NKp44, NKp30, NKp46, NKG2D,and/or NKG2C. In some embodiments, the transmembrane domain or fragmentthereof of a CSR as described herein comprises a transmembrane domainselected from the transmembrane domain of CD28 and CD8.

In one embodiment, the first CSR and/or second CSR further comprises thefirst five amino acids of CD3 zeta (CD3C) (SEQ ID NOs: 7 and 6). CD3 isa T cell co-receptor that facilitates T lymphocytes activation whensimultaneously engaged with the appropriate co-stimulation (e.g.,binding of a co-stimulatory molecule). A CD3 complex consists of 4distinct chains; mammalian CD3 consists of a CD3y chain, a CD35 chain,and two CD3s chains. These chains associate with the T cell receptor(TCR) and CD3ζ to form a complex that generates an activation signal inT lymphocytes. In alternative embodiments, the first CSR and/or secondCSR further comprises full length CD3ζ, truncated CD3ζ, or a fragmentthereof that permit function in the context of the TCR complex.

In one embodiment, the first CSR and/or second CSR further comprises afluorescent protein. In one embodiment, the first CSR and the second CSRexpress different color fluorescent proteins (i.e., the first CSRexpresses a green fluorescent protein and the second CSR expresses a redfluorescent protein). Non-limiting examples of green fluorescentproteins include GFP, eGFP, Emerald, Superfolder GFP, Azami Green,mWasabi, TagGFP, TurboGFP, AcGFP, ZsGreen, and T-Sapphire. Non-limitingexamples of blue fluorescent proteins include eBFP, eBFP2, Azurite, andmTagBFP. Non-limiting examples of cyan fluorescent proteins includeeCFP, mECFP, Cerulean, mTurquoise, CyPet, AmCyanl, Midori-Ishi Cyan,TagCFP, and mTFP!. Non-limiting examples of yellow fluorescent proteinsinclude eYFP, Topaz, Venus, mCitrine, YPet, TagYFP, PhiYFP, ZsYellowl,and mBanana. Non-limiting examples of orange fluorescent proteinsinclude Kusabira Orange, Kxisabira Qrange2, mOrange, mOrange2, dTomato,dTomato-Tandera, TagRFP, TagRFP-T, DsRed, DsRed2, DsRed-Express,DsRed-Monomer, and mTangerine. Non-limiting examples of red fluorescentproteins include mRuby, mApple, mStrawberry, AsRed2, JRed, mCherry,HcRedl, mRasberry, dKeima-Tandem, HcRed-Tandem, mPlum, and AQ143. In oneembodiment, the fluorescent protein comprised in the first CSR andsecond CSR is used to determine if an aAPC is expressing the first CSRand/or second CSR, for example by FACS sorting or microscopy analysis.

In one embodiment, the first CSR is a CD3 (OKT3) CSR, which comprisesthe CD8 hinge region and TM domain, the first 5 amino acids of CD3ζ, T2Alinker, and GFP and comprises a sequence selected from SEQ ID NO: 1 or10. In one embodiment, the second CSR is a CD28 CSR (9.3), whichcomprises the CD28 hinge region, TM domain, and intracellular domain,T2A linker, and mCherry and comprises a sequence of SEQ ID NO: 19.

In one embodiment, the first CSR sequence corresponds to the sequence ofSEQ ID NO: 1 or 10; or comprises the sequence of SEQ ID NO: 1 or 10; orcomprises a sequence with at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% or greater sequenceidentity to the sequence of SEQ ID NO: 1 or 10.

In one embodiment, the second CSR sequence corresponds to the sequenceof SEQ ID NO: 19; or comprises the sequence of SEQ ID NO: SEQ ID NO: 19;or comprises a sequence with at least 80%, at least 85%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% or greater sequenceidentity to the sequence of SEQ ID NO: 19.

A “T cell receptor” (TCR) refers to a heterodimeric receptor moleculefound on the surface of a T cell that recognizes and binds an antigenbound to/displayed upon the MHC or an APC. Binding of MHC-displayedantigen by the TCR initiates signal transduction by the TCR necessaryfor activation of the T cell. For a T cell to be fully activated, the Tcell must be co-stimulated (e.g., receiving simultaneous first andsecond signaling via binding of an antigen-specific and antigennon-specific molecule, respectively).

In one embodiment, the first CSR comprises an antibody orantigen-binding domain thereof that specifically binds to CD28, and thesecond CSR comprises an antibody or antigen-binding domain thereof thatspecifically binds to CD3. Binding of CD28 and CD3 by the anti-CD28 andanti-CD3 domains stimulate T cells to proliferate and/or activate. Inanother embodiment, the first CSR comprises an antibody orantigen-binding domain thereof specific for binding to 4-1BBL; and thesecond CSR comprises an antibody or antigen-binding domain thereofspecific for binding to OX40L. In another embodiment, the first CSRcomprises a natural ligand that binds specifically to 4-1BBL, and thesecond CSR comprised a natural ligand that binds specifically to OX40L.It is contemplated herein that any other known T cell receptors and/orco-stimulatory molecules can be used to generate aAPC's as describedherein.

CD28 is a receptor protein expressed on a T cell surface that provides aco-stimulatory signal for T cell activation. As noted above, engagementof CD28 and CD3 by their ligands promote T cell proliferation. CD28natural ligands include CD80 (B7-1) and CD86 (B7-2). CD28 sequences areknown for a number of species, e.g., human CD28 (NCBI Gene ID: 940) andmRNA (NCBI Ref Seq NM_001243077.1). CD28 can refer to human CD28,including naturally occurring variants and alleles thereof. In someembodiments of, e.g., in veterinary applications, CD28 can refer to theCD28 of, e.g., dog, cat, cow, horse, pig, and the like. Homologs and/ororthologs of human CD28 are readily identified for such species by oneof skill in the art, e.g., using the NCBI ortholog search function orsearching available sequence data for a given species for sequencesimilar to a reference CD28 sequence.

CD3 is a T cell co-receptor found on the surface of a T cell. CD3sequences are known for a number of species, e.g., human CD3e (NCBI GeneID: 916) and mRNA (NCBI Ref Seq NM_000733.3). CD3 can refer to humanCD3, including naturally occurring variants, molecules, and allelesthereof In some embodiments of any of the aspects, e.g., in veterinaryapplications, CD3 can refer to the CD3 of, e.g., dog, cat, cow, horse,pig, and the like. Homologs and/or orthologs of human CD3 are readilyidentified for such species by one of skill in the art, e.g., using theNCBI ortholog search function or searching available sequence data for agiven species for sequence similar to a reference CD3 sequence.

4-1BBL is a type 2 transmembrane glycoprotein belonging to the TNFR/TNFligand superfamily. 4-1BBL is a co-stimulatory ligand that bindsreceptor 4-1BB (CD137) expressed on T cell. 4-1BBL is expressed onprofessional APCs including dendritic cells, macrophages, and activatedB cells. 4-1BBL sequences are known for a number of species, e.g., human4-1BBL, also known as TNFSF9 (NCBI Gene ID: 8744) and mRNA (NCBI Ref SeqNM_00381 1.3). 4-1BBL can refer to human 4-1BBL, including naturallyoccurring variants, molecules, and alleles thereof. In some embodimentsof any of the aspects, e.g., in veterinary applications, 4-1BBL canrefer to the 4-1BBL of, e.g., dog, cat, cow, horse, pig, and the like.Homologs and/or orthologs of human 4-1BBL are readily identified forsuch species by one of skill in the art, e.g., using the NCBI orthologsearch function or searching available sequence data for a given speciesfor sequence similar to a reference 4-1BBL sequence.

OX40L is a natural ligand for the CD 134 receptor, OX40. OX40L isexpressed on APCs, and activated T cells, among others. OX40 and OX40Lare both expressed at higher levels after antigen presentation to a Tcell. CD28 ligation also induces OX40 and OX40L expression. OX40Lsequences are known for a number of species, e.g., human OX40L, alsoknown as TNFSF4 (NCBI Gene ID: 7292) and mRNA (NCBI Ref SeqNM_001297562.1). OX40L can refer to human OX40L, including naturallyoccurring variants, molecules, and alleles thereof In some embodimentsof any of the aspects, e.g., in veterinary applications, OX40L can referto the OX40L of, e.g., dog, cat, cow, horse, pig, and the like. Homologsand/or orthologs of human OX40L are readily identified for such speciesby one of skill in the art, e.g., using the NCBI ortholog searchfunction or searching available sequence data for a given species forsequence similar to a reference OX40L sequence.

In one embodiment, an aAPC as described herein is engineered to lack anexpressible low density lipoprotein receptor (LDLR) gene. In anotherembodiment, an aAPC comprises a deletion in the native LDLR-encodingnucleic acid sequence. In another embodiment, an aAPC comprises adeletion of the native LDLR-encoding nucleic acid sequence, such thatthe cell does not express a functional LDLR gene product. The lack of afunction LDLR can increase the capacity of the aAPC to take up alentivirus (described herein below).

LDLR is a cell surface protein that mediates endocytosis of low densitylipoprotein. LDLR sequences are known for a number of species, e.g.,human LDLR, (NCBI Gene ID: 3949) and mRNA (NCBI Ref Seq NM_000527.4).LDLR can refer to human LDLR, including naturally occurring variants,molecules, and alleles thereof. In some embodiments, e.g., in veterinaryapplications, LDLR can refer to the LDLR of, e.g., dog, cat, cow, horse,pig, and the like. Homologs and/or orthologs of human LDLR are readilyidentified for such species by one of skill in the art, e.g., using theNCBI ortholog search function or searching available sequence data for agiven species for sequence similar to a reference LDLR sequence.

One skilled in the art can engineer an aAPC to lack an expressible LDLRgene using standard techniques. In one embodiment, therecently-discovered CRISPR-associated (Cas) system, such as CRISPR-Cas9,can be used for genome-editing. CRISPR-Cas technology for editing ofgenomes is fully described in Doudna, J A, and Charpentier, E. Science,346: 6213, 2014, which is incorporated by reference herein in itsentirety. This is a practicable, convenient and flexible method of geneediting, and a number of adaptations and improvements on the basictechnology are now in common use.

As described herein, a deletion of an entire gene, or fragment thereofcan be introduced by utilizing the CRISPR/Cas system. Non-limitingexemplary CRISPR-Cas9 methods for inducing the loss of a functional LDLRgene are described in Example 1.

In alternative embodiments, mutations to delete LDLR coding sequence orotherwise mutate the gene to either eliminate expression of the proteinor eliminate expression of functional LDLR protein can be introduced byutilizing TALENs or ZFN technology, which are known in the art. Methodsof engineering nucleases to achieve a desired sequence specificity areknown in the art and are described, e.g., in Kim (2014); Kim (2012);Belhaj et al. (2013); Urnov et al. (2010); Bogdanove et al. (2011);Jinek et al. (2012) Silva et al. (2011); Ran et al. (2013); Carlson etal. (2012); Guerts et al. (2009); Taksu et al. (2010); and Watanabe etal. (2012); each of which is incorporated by reference herein in itsentirety.

In alternate embodiments, LDLR gene expression can be depleted via othertechniques known in the field. In some embodiments, an agent thatinhibits LDLR gene or gene product expression is an inhibitory nucleicacid. As used herein, “inhibitory nucleic acid” refers to a nucleic acidmolecule, e.g., double-stranded RNAs (dsRNAs), inhibitory RNAs (iRNAs),and the like which can inhibit the expression of a target gene.Inhibitory nucleic acid technology is known to those of ordinary skillin the art and is more fully described in, e.g., Wilson, R C, andDoudna, J A. (2013) Annual Review of Biophysics 42(217-239) andreferences cited therein. In another embodiment, the agent is a nucleicacid that encodes a protein that specifically bind to and inhibits LDLRfunction. A non-limiting example of a protein that binds to and inhibitsLDLR function is Proprotein convertase subtilisin/kexin type 9 (PCSK9).

One can evaluate the lack of expressible LDLR gene or LDLR geneexpression, for example by RT-PCR, northern blotting, western blotting,ELISA, or immunohistochemistry. To evaluate the presence of a functionalLDLR gene, one can evaluate whether LDLR is capable of mediatingendocytosis of low density lipoprotein using standard assays.

In one embodiment, the aAPC is viable but non-dividing. aAPCs can berendered non-dividing, for example by gamma irradiation or mitomycintreatment. See, e.g., Llames et al. Tissue Eng. Part B Rev. 21:345-353(2015) and references cited therein. As one example, the aAPCs can betreated with gamma rays in the range of 3,000 to 3,600 rads.

One skilled in the art can assess the cellular viability of an aAPCusing various assays, for example by dye exclusion assays using, e.g.,trypan blue, eosin or propidium iodide. One can assess whether an aAPCis dividing using proliferation assays known in the art, for exampleusing a [³H] thymidine incorporation assay.

In some embodiments, aAPC are frozen, and thawed when needed, retainingfunction as an aAPC. In some embodiments, the frozen aAPC is stored atat least −20° C. In some embodiments, frozen aAPC are stored at at least−80° C. In some embodiments, frozen aAPC are stored in liquid nitrogen.In some embodiments of any aspect, frozen aAPC can be stored for atleast 1 day, at least 2 days, at least 3 days, at least 4 days, at least5 days, at least 6 days, at least 1 week, at least 2 weeks, at least 3weeks, at least 1 month, at least 2 months, at least 3 months, at least4 months, at least 5 months, at least 6 months, at least 7 months, atleast 8 months, at least 9 months, at least 10 months, at least 11months, at least 1 year, at least 2 years, at least 3 years, at least 4years, at least 5 years, or more. In one embodiment, frozen aAPCs arestored in liquid nitrogen for more than 2 years.

In one embodiment, aAPC are slow thawed, e.g., placed in ice and allowedto thaw without any intervention. In one embodiment, aAPC are quickthawed, e.g., the frozen aAPC are placed in a water bath at 37° C. tothaw, and then placed on ice immediately after thawing is complete. Asused herein, “a functional aAPC” refers to an aAPC that functions likeand resembles an aAPC prior to being frozen (i.e., the thawed aAPCexpresses the first CSR and second CSR at the same level as prior tobeing frozen). In one embodiment of any aspect, a thawed aAPC does notproliferate.

In one embodiment, the aAPC further comprises a T cell target molecule.As used herein, a “T cell target molecule” is a ligand for a T cellreceptor, e.g., a naturally-occurring T cell receptor, or an engineeredT cell receptor, e.g., a CAR. A T cell target molecule will activate a Tcell, provided appropriate co-stimulatory signals. In this context, theT cell target molecule can be a full length target polypeptide, i.e., asit occurs in vivo or native to the genome, or a fragment thereof thatbinds the T cell receptor and promotes proliferation and/or activationof the T cell (given the appropriate co-stimulatory signals).

In one embodiment, the T cell target molecule expressed on the surfaceof an aAPC includes, but is not limited to CD19, BCMA, SLAMF7, EGFR orEGFR variant III. In one embodiment, the T cell target molecule is CD19. In addition, it is contemplated that any T cell target moleculeexpressed on the aAPC can be targeted by a chimeric antigen receptorCAR-that is, when an aAPC is engineered to express a T cell targetmolecule, that molecule can be any molecule that binds theligand-binding moiety of a T cell receptor, e.g., a CAR or other T cellreceptor. In one embodiment, the T cell target molecule expressed on theaAPC is constitutively expressed. In another embodiment, the T celltarget molecule expressed on the aAPC is expressed in a facultativemanner. One skilled in the art would be capable of engineering an aAPCto express a T cell target molecule using known techniques.

In one embodiment, the expression of a T cell target molecule expressedon the surface of the aAPC will facilitate the recognition of the aAPCby a T cell or CAR T cell. In this embodiment, the presence of the Tcell target molecule on the aAPC provides a mechanism for eliminatingthe aAPC via T cell-mediated mechanisms.

In one embodiments of any aspect, the aAPC is engineered from a humancell. In one embodiments, the aAPC is engineered from an erthromyeloidcell. In one embodiment, the erthromyeloid cell is a K562 cell. Inanother embodiment, an aAPC is engineered from a cell of myeloid origin.Non-limiting examples of cells of myeloid origin include megakaryocytes,thrombocytes, erythrocytes, mast cells, myeloblasts, basophils,neutrophils, eosinophils, monocytes, macrophages, dendritic cells, andplatelets. In another embodiment, an aAPC is engineered from a celllacking HLA expression (e.g., an immature hematopoietic cell), or a cellthat has been engineered to lack functional HLA via gene editing (e.g.,CRISPR). An aAPC can be engineered from an non-adherent cell or anadherent cell.

K562 is an immortalized myelogenous leukemia cell line. The line is ofthe erythroleukemia type, derived from a patient presenting with chronicmyelogenous leukemia in blast crisis. While other cells can beengineered to generate aAPCs as described herein, K562 cells are wellsuited for use as an aAPC as they do not express majorhistocompatibility complex molecules, and therefore would not provokeallogeneic responses. Further, K562 cells express adhesion moleculesthat enhance T cell-aAPC interactions. K562 cells are commerciallyavailable, for example from American Type Culture Collection (ATCC),Manassas, Virginia, product ATCC CCL-243. Standard cell cultureprotocols and techniques known in the art are used to maintain K562cells in culutre.

In one embodiment, the first CSR and second CSR are expressed in theK562 cell via lentiviral expression, e.g., retroviral vector.Retroviruses, such as lentiviruses, provide a convenient platform fordelivery of nucleic acid sequences encoding a gene, or chimeric gene ofinterest. A selected nucleic acid sequence can be inserted into a vectorand packaged in retroviral particles using techniques known in the art.The recombinant virus can then be isolated and delivered to cells, e.g.in vitro or ex vivo. Retroviral systems are well known in the art andare described in, for example, U.S. Pat. No. 5,219,740; Kurth andBannert (2010) “Retroviruses: Molecular Biology, Genomics andPathogenesis” Calster Academic Press (ISBN:978-1-90455-55-4); and Hu andPathak Pharmacological Reviews 2000 52:493-512; which are incorporatedby reference herein in their entirety. Components of lentiviral systemfor efficient DNA delivery can be purchased from OriGene; Rockville, MD.

In one aspect provided herein is a method for expanding and/oractivating a T cell or population thereof, comprising contacting any ofthe aAPC described herein with a T cell, or population thereof.

In one aspect provided herein is a method for treating cancer, a plasmacell disease or disorder, or an autoimmune disease or disordercomprising contacting an aAPC as described above with a CAR T cell orpopulation thereof, thereby activating the CAR T cell, and administeringthe activated CAR T cell to a subject in need thereof

T cell activation occurs through simultaneous engagement of the T cellreceptor and co-stimulatory molecules (i.e., CD3 and CD28). This resultsin the activation of downstream signaling pathways (e.g., PI3Ksignaling), and eventual immune response (involving cytokineproduction). Following activation, a T cell expresses a variety ofproteins (also known as markers), including, but not limited to CD69,CD71, CD25, and HLA-DR. In addition, an activated T cell has an alteredcell surface protein glycosylation profile.

T cells to be contacted with an aAPC can be obtained using standardtechniques known in the field. For example, T cells can be isolated fromperipheral blood taken from a donor or patient. T cells can be isolatedfrom a mammal. Preferably, T cells are isolated from a human. Aordinarily skilled person can generate CAR T cells to be contacted withan aAPC using standard techniques. Briefly, T cells isolated fromperipheral blood of a patient are engineered to express a chimericantigen receptor on their surface using viral or non-viral vectors. Inone embodiment, the T cells can be isolated from a healthy subject. Inantoher embodiment, the T cells can be isolated from a patient havingbeen diagnosed with a disease or disorder.

In one embodiment, the contacting of an aAPC and T cell or CAR T celloccurs in vitro. In another embodiment, the contacting occurs insuspension. If an aAPC is engineered from an adherent cell, the adherentaAPC will be contacted by a T cell in suspension. In another embodiment,an aAPC is contacted with a T cell or CAR T cell for at least 4 days,for at least 5 days, for at least 6 days, for at least 7 days, for atleast 8 days, for at least 9 days, or more. In a preferred embodiment ofany aspect, the contact of the aAPC and T cell or CAR T cell iscontinuous for the duration of the contact.

In one embodiment of any aspect, contacting an aAPC with a T cell or CART cell results in the expansion of the T cell or CAR T cell population.In one embodiment, following contact by an aAPC, the T cell or CAR Tcell population will undergo 5-7 doublings in 10 days. In oneembodiment, following contact by an aAPC, the T cell or CAR T cellpopulation increases at least 1-fold, at least 2-fold, at least 3-fold,at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, atleast 9-fold, or at least 10-fold or more. One skilled in the art willbe capable of measuring T cell or CAR T cell growth, for example, via aT cell expansion curve. Briefly, T cells or CAR T cells are seeded atday 0 with an aAPC or population thereof Total T cell or CAR T cellnumbers and mean T cell or CAR T cell volumes are measured at days 3, 5,7, and 9 using, e.g., a Multisizer™ 3 Coulter Counter (Beckman Coulter).Cellular viability can be determined by staining, e.g., with AcridineOrange/Propidiumlodide exclusion dye using a Luna-FL™Cell Counter (LogosBiosystems).

One skilled in the art will be capable of determining if a T cell or CART cell has become activated following contact with an aAPC. For example,one can use a Proliferative Capacity assay (CFSE dilution and absolute Tcell or CAR T cell numbers are assessed by FACS usingfluorescently-labeled counting beads), a Cytokine Production assay(10-Plex Luminex Assays using cytokine levels as a readout), aTarget-cell Killing Capacity assay (Bioluminescence analysis of targetcells in vitro or animal model system to track both tumor and engineeredT cells infused in immunodeficient mice), and/or a Cell DegranulationAnalysis (CD107a release assay in response to target cells as measuredby FACS). One skilled in the art can additionally determine if a T cellis activated by assessing the markers present on the T cell surface, orby examining the glycosylation profile of the cell surface.

In one embodiment of any aspect, a subject is administered activated CART cells that are engineered to express an XCAR, wherein X represents atumor antigen (or cancer associated antigen) as described herein that isexpressed on a subject's tumor cell.

In one embodiment, a subject is administered activated CAR T cells thatare engineered to express an XCAR described herein, wherein the cancercells express X on their surface. In one embodiment, X is expressed onboth normal cells and cancers cells, but is expressed at lower levels onnormal cells. In one embodiment, the method further comprises selectinga CAR that binds X with an affinity that allows the XCAR to recognizeand kill the cancer cells expressing X but less than 30%, 25%, 20%, 15%,10%, 5% or less of the normal cells expressing X are killed. Suchactivity can be evaluated for example in a cell killing assay such as achromium-51 release cytotoxicity assay or a similar fluorescence-basedassay. In one embodiment, the selected CAR has an antigen binding domainthat has a binding affinity KD of 10⁻⁴ M to 10⁻⁸ M, e.g., 10⁻⁵ M to 10⁻⁷M, e.g., 10⁻⁶ M or 10⁻⁷ M, for the target antigen.

Described herein is a type of cellular therapy in which activated CARTcells are infused to a recipient in need thereof The infused cell isable to kill tumor cells in the recipient. Unlike antibody therapies,CAR T cells, are able to replicate in vivo, resulting in long-termpersistence that can lead to sustained tumor control. In variousembodiments, activated CAR T cells administered to the patient persistin the patient for at least four months, five months, six months, sevenmonths, eight months, nine months, ten months, eleven months, twelvemonths, thirteen months, fourteen month, fifteen months, sixteen months,seventeen months, eighteen months, nineteen months, twenty months,twenty-one months, twenty-two months, twenty-three months, two years,three years, four years, or five years after administration of the Tcell to the patient.

In one embodiment, activated CAR T cells are administered to a subjectwho has cancer. In one embodiment, the subject has been diagnosed withcancer. In one embodiment, the cancer is leukemia, lymphoma, multiplemyeloma, or a solid tumor.

With respect to leukemia, non-limiting examples of leukemia includeacute myeloid leukemia (AML), Chronic myeloid leukemia (CML), Acutelymphocytic leukemia (ALL), and Chronic lymphocytic leukemia (CLL). Inone embodiment, the cancer is ALL or CLL.

With respect to lymphoma, non-limiting examples of lymphoma includeDiffuse large B-cell lymphoma (DLBCL), Follicular lymphoma, Smalllymphocytic lymphoma (SLL), Mantle cell lymphoma (MCL), Marginal zonelymphomas, Burkitt lymphoma, hairy cell leukemia. (HCL).

In one embodiment, the cancer is DLBCL or Follicular lymphoma.

With respect to solid tumors, non-limiting examples of solid tumorsinclude Adrenocortical Tumor, Alveolar Soft Part Sarcoma,Chondrosarcoma, Colorectal Carcinoma, Desmoid Tumors, Desmoplastic SmallRound Cell Tumor, Endocrine Tumors, Endodermal Sinus Tumor, EpithelioidHemangioendothelioma, Ewing Sarcoma, Germ Cell Tumors, Giant Cell Tumorof Bone and Soft Tissue, Hepatoblastoma, Hepatocellular Carcinoma,Melanoma, Nephroma, Neuroblastoma, Non-Rhabdomyosarcoma Soft TissueSarcoma (NRSTS), Osteosarcoma, Paraspinal Sarcoma, Renal Cell Carcinoma,Retinoblastoma, Rhabdomyosarcoma, Synovial Sarcoma, and Wilms Tumor.Solid tumors can be found in bones, muscles, or organs, and can besarcomas or carinomas.

It is contemplated that CAR T cells activated following contact by anyof the aAPC described herein can be used to treat all types of cancers,including cancers not listed herein. Effective treatment requires thatone or more tumor antigens are known or identified for the cancer inquestion, and that an antigen-binding domain specific for the tumorantigen(s) is identified and cloned. When these conditions are met, oneof skill in the art can prepare a CAR, introduce it to a T cell, andactivate those T cells using an aAPC as described herein. Theidentification of a tumor antigen present on a tumor cell can befacilitated by assessing a tumor sample for known tumor antigens, e.g.,using fluorescence microscopy or FACS with a panel of anti-tumor antigenantibodies.

Administration

In some embodiments, the methods described herein relate to treating asubject having or diagnosed as having cancer by administering activatedCAR T cells. As used herein, a “condition” refers to a cancer, a plasmacell disease or disorder, or an autoimmune disease or disorder. Subjectshaving a condition can be identified by a physician using currentmethods of diagnosing the condition. Symptoms and/or complications ofthe condition, which characterize these conditions and aid in diagnosisare well known in the art and include but are not limited to, fatigue,persistent infections, and persistent bleeding. Tests that may aid in adiagnosis of, e.g. the condition, but are not limited to, bloodscreening and bone marrow testing, and are known in the art for a givencondition. A family history for a condition, or exposure to risk factorsfor a condition can also aid in determining if a subject is likely tohave the condition or in making a diagnosis of the condition.

The CAR T cell compositions described herein can be administered to asubject having or diagnosed as having a condition. In some embodiments,the methods described herein comprise administering an effective amountof activated CAR T cells described herein to a subject in order toalleviate a symptom of the condition. As used herein, “alleviating asymptom of the condition” is ameliorating any condition or symptomassociated with the condition. As compared with an equivalent untreatedcontrol, such reduction is by at least 5%, 10%, 20%, 40%, 50%, 60%, 80%,90%, 95%, 99% or more as measured by any standard technique. A varietyof means for administering the compositions described herein to subjectsare known to those of skill in the art. In one embodiment, the CAR Tcell compositions described herein are administered systemically orlocally. In a preferred embodiment, the compositions are administeredintravenously. In another embodiment, the compositions are administeredat the site of a tumor.

The term “effective amount” as used herein refers to the amount ofactivated CAR T cells needed to alleviate at least one or more symptomof the disease or disorder, and relates to a sufficient amount of thecell preparation or composition to provide the desired effect. The term“therapeutically effective amount” therefore refers to an amount ofactivated CAR T cells that is sufficient to provide a particularanti-condition effect when administered to a typical subject. Aneffective amount as used herein, in various contexts, would also includean amount sufficient to delay the development of a symptom of thedisease, alter the course of a symptom disease (for example but notlimited to, slowing the progression of a condition), or reverse asymptom of the condition. Thus, it is not generally practicable tospecify an exact “effective amount”. However, for any given case, anappropriate “effective amount” can be determined by one of ordinaryskill in the art using only routine experimentation.

Effective amounts, toxicity, and therapeutic efficacy can be evaluatedby standard pharmaceutical procedures in cell cultures or experimentalanimals. The dosage can vary depending upon the dosage form employed andthe route of administration utilized. The dose ratio between toxic andtherapeutic effects is the therapeutic index and can be expressed as theratio LD50/ED50. Compositions and methods that exhibit large therapeuticindices are preferred. A therapeutically effective dose can be estimatedinitially from cell culture assays. Also, a dose can be formulated inanimal models to achieve a circulating plasma concentration range thatincludes the IC50 (i.e., the concentration of activated CAR T cells,which achieves a half-maximal inhibition of symptoms) as determined incell culture, or in an appropriate animal model. Levels in plasma can bemeasured, for example, by high performance liquid chromatography. Theeffects of any particular dosage can be monitored by a suitablebioassay, e.g., assay for bone marrow testing, among others. The dosagecan be determined by a physician and adjusted, as necessary, to suitobserved effects of the treatment.

In one aspect of the invention, the technology described herein relatesto a pharmaceutical composition comprising activated CAR T cells asdescribed herein, and optionally a pharmaceutically acceptable carrier.The active ingredients of the pharmaceutical composition at a minimumcomprise activated CAR T cells as described herein. In some embodiments,the active ingredients of the pharmaceutical composition consistessentially of activated CAR T cells as described herein. In someembodiments, the active ingredients of the pharmaceutical compositionconsist of activated CAR T cells as described herein. Pharmaceuticallyacceptable carriers for cell-based therapeutic formulations includesaline and aqueous buffer solutions, Ringer's solution, and serumcomponent, such as serum albumin, HDL and LDL. The terms such as“excipient,” “carrier,” “pharmaceutically acceptable carrier” or thelike are used interchangeably herein.

In some embodiments, the pharmaceutical composition comprising activatedCAR T cells as described herein can be a parenteral dose form. Sinceadministration of parenteral dosage forms typically bypasses thepatient's natural defenses against contaminants, the components apartfrom the CAR T cells themselves are preferably sterile or capable ofbeing sterilized prior to administration to a patient. Examples ofparenteral dosage forms include, but are not limited to, solutions readyfor injection, dry products ready to be dissolved or suspended in apharmaceutically acceptable vehicle for injection, suspensions ready forinjection, and emulsions. Any of these can be added to the activated CART cells preparation prior to administration.

Suitable vehicles that can be used to provide parenteral dosage forms ofactivated CAR T cells as disclosed herein are well known to thoseskilled in the art. Examples include, without limitation: salinesolution; glucose solution; aqueous vehicles including but not limitedto, sodium chloride injection, Ringer's injection, dextrose Injection,dextrose and sodium chloride injection, and lactated Ringer's injection.

Dosage

“Unit dosage form” as the term is used herein refers to a dosage forsuitable one administration. By way of example a unit dosage form can bean amount of therapeutic disposed in a delivery device, e.g., a syringeor intravenous drip bag. In one embodiment, a unit dosage form isadministered in a single administration. In another, embodiment morethan one unit dosage form can be administered simultaneously.

In some embodiments, the activated CAR T cells described herein areadministered as a monotherapy, i.e., another treatment for the conditionis not concurrently administered to the subject.

A pharmaceutical composition comprising the T cells described herein cangenerally be administered at a dosage of 10⁴ to 10⁹ cells/kg bodyweight, in some instances 10⁵ to 10⁶ cells/kg body weight, including allinteger values within those ranges. If necessary, T cell compositionscan also be administered multiple times at these dosages. The cells canbe administered by using infusion techniques that are commonly known inimmunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med.319:1676, 1988).

In certain aspects, it may be desired to administer activated CAR Tcells to a subject and then subsequently redraw blood (or have anapheresis performed), activate T cells therefrom as 5 described herein,and reinfuse the patient with these activated and expanded T cells. Thisprocess can be carried out multiple times every few weeks. In certainaspects, T cells can be activated from blood draws of from 1Occ to 400cc. In certain aspects, T cells are activated from blood draws of 20 cc,30 cc, 40 cc, 50 cc, 60 cc, 70 cc, 80 cc, 90 cc, or 1OOcc.

Modes of administration can include, for example intravenous (i.v.)injection or infusion. The compositions described herein can beadministered to a patient transarterially, intratumorally, intranodally,or intramedullary. In some embodiments, the compositions of T cells maybe injected directly into a tumor, lymph node, or site of infection. Inone embodiment, the CAR T cell compositions are administered into a bodycavity or body fluid (e.g., ascites, pleural fluid, peritoneal fluid, orcerebrospinal fluid).

In a particular exemplary aspect, subjects may undergo leukapheresis,wherein leukocytes are collected, enriched, or depleted ex vivo toselect and/or isolate the cells of interest, e.g., T cells. These T cellisolates can be expanded by contact with an aAPC as described herein,e.g., an aAPC expressing anti-CD28 and anti-CD3 CDRs as described hereinand treated such that one or more CAR constructs of the invention may beintroduced, thereby creating a CAR T cell. Subjects in need thereof cansubsequently undergo standard treatment with high dose chemotherapyfollowed by peripheral blood stem cell transplantation. Following orconcurrent with the transplant, subjects can receive an infusion of theexpanded CAR T cells. In one embodiment, expanded cells are administeredbefore or following surgery.

In some embodiments, lymphodepletion is performed on a subject prior toadministering one or more CAR T cell as described herein. In suchembodiments, the lymphodepletion can comprise administering one or moreof melphalan, Cytoxan, cyclophosphamide, and fludarabine.

The dosage of the above treatments to be administered to a patient willvary with the precise nature of the condition being treated and therecipient of the treatment. The scaling of dosages for humanadministration can be performed according to art- accepted practices.

In some embodiments, a single treatment regimen is required. In others,administration of one or more subsequent doses or treatment regimens canbe performed. For example, after treatment biweekly for three months,treatment can be repeated once per month, for six months or a year orlonger. In some embodiments, no additional treatments are administeredfollowing the initial treatment.

The dosage of a composition as described herein can be determined by aphysician and adjusted, as necessary, to suit observed effects of thetreatment. With respect to duration and frequency of treatment, it istypical for skilled clinicians to monitor subjects in order to determinewhen the treatment is providing therapeutic benefit, and to determinewhether to administer further cells, discontinue treatment, resumetreatment, or make other alterations to the treatment regimen. Thedosage should not be so large as to cause adverse side effects, such ascytokine release syndrome. Generally, the dosage will vary with the age,condition, and sex of the patient and can be determined by one of skillin the art. The dosage can also be adjusted by the individual physicianin the event of any complication.

Combinational Therapy

The activated CAR T cells described herein can be used in combinationwith other known agents and therapies. Administered “in combination,” asused herein, means that two (or more) different treatments are deliveredto the subject during the course of the subject's affliction with thedisorder, e.g., the two or more treatments are delivered after thesubject has been diagnosed with the disorder and before the disorder hasbeen cured or eliminated or treatment has ceased for other reasons. Insome embodiments, the delivery of one treatment is still occurring whenthe delivery of the second begins, so that there is overlap in terms ofadministration. This is sometimes referred to herein as “simultaneous”or “concurrent delivery.” In other embodiments, the delivery of onetreatment ends before the delivery of the other treatment begins. Insome embodiments of either case, the treatment is more effective becauseof combined administration. For example, the second treatment is moreeffective, e.g., an equivalent effect is seen with less of the secondtreatment, or the second treatment reduces symptoms to a greater extent,than would be seen if the second treatment were administered in theabsence of the first treatment, or the analogous situation is seen withthe first treatment. In some embodiments, delivery is such that thereduction in a symptom, or other parameter related to the disorder isgreater than what would be observed with one treatment delivered in theabsence of the other. The effect of the two treatments can be partiallyadditive, wholly additive, or greater than additive. The delivery can besuch that an effect of the first treatment delivered is still detectablewhen the second is delivered. The activated CAR T cells described hereinand the at least one additional therapeutic agent can be administeredsimultaneously, in the same or in separate compositions, orsequentially. For sequential administration, the CAR-expressing celldescribed herein can be administered first, and the additional agent canbe administered second, or the order of administration can be reversed.The CAR T therapy and/or other therapeutic agents, procedures ormodalities can be administered during periods of active disorder, orduring a period of remission or less active disease. The CAR T therapycan be administered before another treatment, concurrently with thetreatment, post-treatment, or during remission of the disorder.

When administered in combination, the activated CAR T cells and theadditional agent (e.g., second or third agent), or all, can beadministered in an amount or dose that is higher, lower or the same asthe amount or dosage of each agent used individually, e.g., as amonotherapy. In certain embodiments, the administered amount or dosageof the activated CAR T cells, the additional agent (e.g., second orthird agent), or all, is lower (e.g., at least 20%, at least 30%, atleast 40%, or at least 50%) than the amount or dosage of each agent usedindividually. In other embodiments, the amount or dosage of theactivated CAR T cells, the additional agent (e.g., second or thirdagent), or all, that results in a desired effect (e.g., treatment ofcancer) is lower (e.g., at least 20%, at least 30%, at least 40%, or atleast 50% lower) than the amount or dosage of each agent individuallyrequired to achieve the same therapeutic effect. In further embodiments,the activated CAR T cells described herein can be used in a treatmentregimen in combination with surgery, chemotherapy, radiation, an mTORpathway inhibitor, immunosuppressive agents, such as cyclosporin,azathioprine, methotrexate, mycophenolate, and FK506, antibodies, orother immunoablative agents such as CAMPATH, anti-CD3 antibodies orother antibody therapies, cytoxin, fludarabine, rapamycin, mycophenolicacid, steroids, FR901228, cytokines, or a peptide vaccine, such as thatdescribed in Izumoto et al. 2008 J Neurosurg 108:963- 971.

In one embodiment, the activated CAR T cells described herein can beused in combination with a checkpoint inhibitor. Exemplary checkpointinhibitors include anti-PD- 1 inhibitors (Nivolumab, MK-3475,Pembrolizumas, Pidilizumab, AMP-224, AMP-514), anti-CTLA4 inhibitors(Ipilimumab and Tremelimumab), anti-PDL1 inhibitors (Atezolizumab,Avelomab, MSB0010718C, MEDI4736, and MPDL3280A), and anti-TIM3inhibitors.

In one embodiment, the activated CAR T cells described herein can beused in combination with a chemotherapeutic agent. Exemplarychemotherapeutic agents include an anthracycline (e.g., doxorubicin(e.g., liposomal doxorubicin)), a vinca alkaloid (e.g., vinblastine,vincristine, vindesine, vinorelbine), an alkylating agent (e.g.,cyclophosphamide, decarbazine, melphalan, ifosfamide, temozolomide), animmune cell antibody (e.g., alemtuzamab, gemtuzumab, rituximab,tositumomab), an antimetabolite (including, e.g., folic acidantagonists, pyrimidine analogs, purine analogs and adenosine deaminaseinhibitors (e.g., fludarabine)), an mTOR inhibitor, a TNFRglucocorticoid induced TNFR related protein (GITR) agonist, a proteasomeinhibitor (e.g., aclacinomycin A, gliotoxin or bortezomib), animmunomodulator such as thalidomide or a thalidomide derivative (e.g.,lenalidomide). General chemotherapeutic agents considered for use incombination therapies include anastrozole (Arimidex®), bicalutamide(Casodex®), bleomycin sulfate (Blenoxane®), busulfan (Myleran®),busulfan injection (Busulfex®), capecitabine (Xeloda®),N4-pentoxycarbonyl-5- deoxy-5-fluorocytidine, carboplatin (Paraplatin®),carmustine (BiCNU®), chlorambucil (Leukeran®), cisplatin (Platinol®),cladribine (Leustatin®), cyclophosphamide (Cytoxan® or Neosar®),cytarabine, cytosine arabinoside (Cytosar-U®), cytarabine liposomeinjection (DepoCyt®), dacarbazine (DTIC-Dome®), dactinomycin(Actinomycin D, Cosmegan), daunorubicin hydrochloride (Cerubidine®),daunorubicin citrate liposome injection (DaunoXome®), dexamethasone,docetaxel (Taxotere®), doxorubicin hydrochloride (Adriamycin®, Rubex®),etoposide (Vepesidt), fludarabine phosphate (Fludara®), 5-fluorouracil(Adrucil®, Efudex®), flutamide (Eulexin®), tezacitibine, Gemcitabine(difluorodeoxycitidine), hydroxyurea (Hydrea®), Idarubicin (Idamycin®),ifosfamide (IFEX®), irinotecan (Camptosar®), L-asparaginase (ELSPARED),leucovorin calcium, melphalan (Alkeran®), 6-mercaptopurine(Purinethol®), methotrexate (Folex®), mitoxantrone (Novantrone®),mylotarg, paclitaxel (Taxol®), phoenix (Yttrium90/MX-DTPA), pentostatin,polifeprosan 20 with carmustine implant (Gliadel®), tamoxifen citrate(Nolvadex®), teniposide (Vumon®), 6-thioguanine, thiotepa, tirapazamine(Tirazone®), topotecan hydrochloride for injection (Hycamptin®),vinblastine (Velban®), vincristine (Oncovin®), and vinorelbine(Navelbine®). Exemplary alkylating agents include, without limitation,nitrogen mustards, ethylenimine derivatives, alkyl sulfonates,nitrosoureas and triazenes): uracil mustard (Aminouracil Mustard®,Chlorethaminacil®, Demethyldopan®, Desmethyldopan®, Haemanthamine ®,Nordopan®, Uracil nitrogen mustard®, Uracillost®, Uracilmostaza®,Uramustin®, Uramustine®), chlormethine (Mustargen®), cyclophosphamide(Cytoxan®, Neosar®, Clafen®, Endoxan®, Procytox®, Revimmune™),ifosfamide (Mitoxana®), melphalan (Alkeran®), Chlorambucil (Leukeran®),pipobroman (Amedel®, Vercyte®), triethylenemelamine (Hemel®, Hexalen®,Hexastat®), triethylenethiophosphoramine, Temozolomide (Temodar®),thiotepa (Thioplex®), busulfan (Busilvex®, Myleran®), carmustine(BiCNU®), lomustine (CeeNU®), streptozocin (Zanosar®), and Dacarbazine(DTIC-Dome ED). Additional exemplary alkylating agents include, withoutlimitation, Oxaliplatin (Eloxatin®); Temozolomide (Temodar® andTemodal®); Dactinomycin (also known as actinomycin-D, Cosmegen®);Melphalan (also known as L-PAM, L-sarcolysin, and phenylalanine mustard,Alkeran®); Altretamine (also known as hexamethylmelamine (HMM),Hexalen®); Carmustine (BiCNU®); Bendamustine (Treanda®); Busulfan(Busulfex R and Myleran®); Carboplatin (Paraplatin®); Lomustine (alsoknown as CCNU, CeeNU®); Cisplatin (also known as CDDP, Platinol® andPlatinol®-AQ); Chlorambucil (Leukeran®); Cyclophosphamide (Cytoxan® andNeosar®); Dacarbazine (also known as DTIC, DIC and imidazolecarboxamide, DTIC-Dome ED); Altretamine (also known ashexamethylmelamine (HMM), Hexalen®); Ifosfamide (Ifex®); Prednumustine;Procarbazine (Matulane®); Mechlorethamine (also known as nitrogenmustard, mustine and mechloroethamine hydrochloride, Mustargen®);Streptozocin (Zanosar®); Thiotepa (also known as thiophosphoamide, TESPAand TSPA, Thioplex®); Cyclophosphamide (Endoxant®, Cytoxan®, Neosar®,Procytoxt®, Revimmunet®); and Bendamustine HC1 (Treanda®). ExemplarymTOR inhibitors include, e.g., temsirolimus; ridaforolimus (formallyknown as deferolimus,(1R,2R,45)-4-[(2R)-2[(1R,95,125,15R,16E,18R,19R,21R,235,24E,26E,28Z,305,325,35R)-1,18-dihydroxy-19,30-dimethoxy-15,17,21,23,29,35- hexamethyl-2,3,10,14,20-pentaoxo-1 1,36-dioxa-4-azatricyclo[30.3.1.04′9]hexatriaconta-16,24,26,28-tetraen-12-yl]propyl]-2-methoxycyclohexyldimethylphosphinate, also known as AP23573 and MK8669, and described inPCT Publication No. WO 03/064383); everolimus (Afinitor® or RADOO1);rapamycin (AY22989, Sirolimus®); simapimod (CAS 164301-51-3);emsirolimus, (5-{2,4-Bis[(35,)-3-methylmorpholin-4-yl]pyrido [2,3-(i]pyrimidin-7-yl }-2-methoxyphenyl)methanol (AZD8055);2-Amino-8-kraw5,-4-(2-hydroxyethoxy)cyclohexyl1-6-(6-methoxy-3-pyridinyl)-4-methyl-pyrido[2,3-LLpyrimidin-7(8H)-one(PF04691502, CAS 1013101-36-4); andN2-[1,4-dioxo-4-[[4-(4-oxo-8-phenyl-4H-1-benzopyran-2-yl)morpholinium-4-yl]methoxy]butyl]-L-arginylglycyl-L-a-aspartylL-serine-(SEQID NO: 39), inner salt (SF1126, CAS 936487-67-1), and XL765. Exemplaryimmunomodulators include, e.g., afutuzumab (available from Roche®);pegfilgrastim (Neulasta®); lenalidomide (CC-5013, Revlimid®);thalidomide (Thalomid®), actimid (CC4047); and IRX-2 (mixture of humancytokines including interleukin 1, interleukin 2, and interferon γ, CAS951209-71-5, available from IRX Therapeutics). Exemplary anthracyclinesinclude, e.g., doxorubicin (Adriamycin® and Rubex®); bleomycin(lenoxane®); daunorubicin (dauorubicin hydrochloride, daunomycin, andrubidomycin hydrochloride, Cerubidine®); daunorubicin liposomal(daunorubicin citrate liposome, DaunoXome®); mitoxantrone (DHAD,Novantrone®); epirubicin (Ellence™); idarubicin (Idamycin®, IdamycinPFS®); mitomycin C (Mutamycin®); geldanamycin; herbimycin; ravidomycin;and desacetylravidomycin. Exemplary vinca alkaloids include, e.g.,vinorelbine tartrate (Navelbine®), Vincristine (Oncovin®), and Vindesine(Eldisine®)); vinblastine (also known as vinblastine sulfate,vincaleukoblastine and VLB, Alkaban-AQ® and Velban®); and vinorelbine(Navelbine®). Exemplary proteosome inhibitors include bortezomib(Velcade®); carfilzomib (PX-171-007,(5)-4-Methyl-N-45)-1-4(5)-4-methyl-14(R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)amino)-1-oxo-3-phenylpropan-2-y1)-2-((5,)-2-(2-morpholinoacetamido)-4-phenylbutanamido)-pentanamide);marizomib (NPT0052); ixazomib citrate (MLN-9708); delanzomib(CEP-18770); and0-Methyl-N-[(2-methyl-5-thiazolyl)carbonyl]-L-seryl-0-methyl-N-[(llS′)-2-[(2R)-2-methyl-2-oxiranyl]-2-oxo-1-(phenylmethy)pethyl]-L-serinamide(ONX-09 12).

One of skill in the art can readily identify a chemotherapeutic agent ofuse (e.g. see Physicians' Cancer Chemotherapy Drug Manual 2014, EdwardChu, Vincent T. DeVita Jr., Jones & Bartlett Learning; Principles ofCancer Therapy, Chapter 85 in Harrison's Principles of InternalMedicine, 18th edition; Therapeutic Targeting of Cancer Cells: Era ofMolecularly Targeted Agents and Cancer Pharmacology, Chs. 28-29 inAbeloffs Clinical Oncology, 2013 Elsevier; and Fischer D S (ed): TheCancer Chemotherapy Handbook, 4th ed. St. Louis, Mosby-Year Book, 2003).

In an embodiment, activated CAR T cells described herein areadministered to a subject in combination with a molecule that decreasesthe a molecule targeting GITR and/or modulating GITR functions, amolecule that decreases the Treg cell population, an mTOR inhibitor, aGITR agonist, a kinase inhibitor, a non-receptor tyrosine kinaseinhibitor, a CDK4 inhibitor, and/or a BTK inhibitor.

Efficacy

The efficacy of activated CAR T cells in, e.g. the treatment of acondition described herein, or to induce a response as described herein(e.g. a reduction in cancer cells) can be determined by the skilledclinician. However, a treatment is considered “effective treatment,” asthe term is used herein, if one or more of the signs or symptoms of acondition described herein is altered in a beneficial manner, otherclinically accepted symptoms are improved, or even ameliorated, or adesired response is induced e.g., by at least 10% following treatmentaccording to the methods described herein. Efficacy can be assessed, forexample, by measuring a marker, indicator, symptom, and/or the incidenceof a condition treated according to the methods described herein or anyother measurable parameter appropriate. Treatment according to themethods described herein can reduce levels of a marker or symptom of acondition, e.g. by at least 10%, at least 15%, at least 20%, at least25%, at least 30%, at least 40%, at least 50%, at least 60%, at least70%, at least 80% or at least 90% or more.

Efficacy can also be measured by a failure of an individual to worsen asassessed by hospitalization, or need for medical interventions (i.e.,progression of the disease is halted). Methods of measuring theseindicators are known to those of skill in the art and/or are describedherein.

Treatment includes any treatment of a disease in an individual or ananimal (some non-limiting examples include a human or an animal) andincludes: (1) inhibiting the disease, e.g., preventing a worsening ofsymptoms (e.g. pain or inflammation); or (2) relieving the severity ofthe disease, e.g., causing regression of symptoms. An effective amountfor the treatment of a disease means that amount which, whenadministered to a subject in need thereof, is sufficient to result ineffective treatment as that term is defined herein, for that disease.Efficacy of an agent can be determined by assessing physical indicatorsof a condition or desired response. It is well within the ability of oneskilled in the art to monitor efficacy of administration and/ortreatment by measuring any one of such parameters, or any combination ofparameters. Efficacy of a given approach can be assessed in animalmodels of a condition described herein, for example treatment of ALL.When using an experimental animal model, efficacy of treatment isevidenced when a statistically significant change in a marker isobserved.

All patents and other publications; including literature references,issued patents, published patent applications, and co-pending patentapplications; cited throughout this application are expresslyincorporated herein by reference for the purpose of describing anddisclosing, for example, the methodologies described in suchpublications that might be used in connection with the technologydescribed herein. These publications are provided solely for theirdisclosure prior to the filing date of the present application. Nothingin this regard should be construed as an admission that the inventorsare not entitled to antedate such disclosure by virtue of priorinvention or for any other reason. All statements as to the date orrepresentation as to the contents of these documents is based on theinformation available to the applicants and does not constitute anyadmission as to the correctness of the dates or contents of thesedocuments.

The description of embodiments of the disclosure is not intended to beexhaustive or to limit the disclosure to the precise form disclosed.While specific embodiments of, and examples for, the disclosure aredescribed herein for illustrative purposes, various equivalentmodifications are possible within the scope of the disclosure, as thoseskilled in the relevant art will recognize. For example, while methodsteps or functions are presented in a given order, alternativeembodiments may perform functions in a different order, or functions maybe performed substantially concurrently. The teachings of the disclosureprovided herein can be applied to other procedures or methods asappropriate. The various embodiments described herein can be combined toprovide further embodiments. Aspects of the disclosure can be modified,if necessary, to employ the compositions, functions and concepts of theabove references and application to provide yet further embodiments ofthe disclosure. Moreover, due to biological functional equivalencyconsiderations, some changes can be made in protein structure withoutaffecting the biological or chemical action in kind or amount. These andother changes can be made to the disclosure in light of the detaileddescription. All such modifications are intended to be included withinthe scope of the appended claims.

Specific elements of any of the foregoing embodiments can be combined orsubstituted for elements in other embodiments. Furthermore, whileadvantages associated with certain embodiments of the disclosure havebeen described in the context of these embodiments, other embodimentsmay also exhibit such advantages, and not all embodiments neednecessarily exhibit such advantages to fall within the scope of thedisclosure.

The technology described herein is further illustrated by the followingexamples which in no way should be construed as being further limiting.

Some embodiments of the technology described herein can be definedaccording to any of the following numbered paragraphs:

-   -   1. An artificial antigen presenting cell (aAPC) comprising:        -   a first chimeric stimulatory receptor (CSR) that binds            specifically with a first co-stimulatory polypeptide; and a            second CSR that binds specifically a second co-stimulatory            polypeptide.    -   2. An artificial antigen presenting cell (aAPC) comprising:        -   a first chimeric stimulatory receptor (CSR) that binds            specifically with CD3; and        -   a second CSR that binds specifically with CD28.    -   3. The aAPC of either paragraph 1 or 2, wherein the aAPC has        been engineered to lack an expressible LDLR gene.    -   4. The aAPC of any of paragraphs 1-3, wherein the aAPC comprises        a deletion in the native

LDLR-encoding nucleic acid sequence.

-   -   5. The aAPC of any of paragraphs 1-3, wherein the aAPC comprises        a deletion of the native

LDLR-encoding nucleic acid sequence.

-   -   6. The aAPC of any of paragraphs 1-5, wherein the aAPC is viable        but non-dividing.    -   7. The aAPC of any of paragraphs 1-6, wherein the aAPC has been        gamma-irradiated.    -   8. The aAPC of any of paragraphs 1-7, further comprising a        T-cell target molecule.    -   9. The aAPC of paragraph 8, wherein the T-cell target molecule        is CD19, BCMA, CD37,

SLAMF7, EGFR, or EGFR variant III.

-   -   10. The aAPC of paragraph 8 or 9, wherein the T-cell target        molecule is CD19.    -   11. The aAPC of any of paragraphs 8-10, wherein the T-cell        target molecule is any molecule that can be targeted by a CAR T        cell.    -   12. The aAPC of any of paragraphs 1-11, wherein the first and        second CSRs are expressed on the cell surface of the aAPC.    -   13. The aAPC of any of paragraphs 1-12, wherein the first and        second CSRs are constitutively expressed.    -   14. The aAPC of any of paragraphs 1-13, wherein the first and        second CSRs are encoded by a first recombinant nucleic acid        sequence and a second recombinant nucleic acid sequence,        respectively.    -   15. The aAPC of paragraph 14, wherein the sequence encoding the        first and/or second CSR is operatively linked to a constitutive        promoter.    -   16. The aAPC of paragraph 15, wherein the constitutive promoter        is EF1-oc.    -   17. The aAPC of any of paragraphs 1-16, wherein the aAPC is a        human cell.    -   18. The aAPC of any of paragraphs 1-17, wherein the aAPC is        engineered from an erythromyeloid cell.    -   19. The aAPC of paragraph 18, wherein the erthromyeloid cell is        a K562 cell.    -   20. The aAPC of any of paragraphs 1-19, wherein the first and        second CSRs bind specifically with human CD3 and CD28,        respectively.    -   21. The aAPC of any of paragraphs 1 and 3-19, wherein the first        CSR binds specifically with 4-1BBL, and the second CSR binds        specifically with OX40L.    -   22. A method of expanding or activating a T cell, the method        comprising contacting the aAPC of any of paragraphs 1-21 with a        T cell.    -   23. A method of treating a cancer, a plasma cell disease or        disorder, or an autoimmune disease or disorder in a subject in        need thereof, the method comprising:        -   contacting the aAPC of any of paragraphs 1-21 with a CAR T            cell, thereby activating the CAR T cell; and        -   administering the activated CAR T cell to the subject.    -   24. The method of paragraph 22 or 23, wherein the contacting        step occurs in vitro.    -   25. The method of paragraph 22 or 23, wherein the contacting        step occurs in suspension.    -   26. A composition comprising the aAPC of any of paragraphs 1-21        and a T cell.    -   27. The composition of paragraph 26, wherein the T cell is a CAR        T cell.    -   28. A composition comprising the activated CAR T cells of the        method of any of paragraphs 23-25, formulated for the treatment        of cancer.    -   29. The composition of paragraph 28, further comprising a        pharmaceutically acceptable carrier.    -   30. The method of any of paragraphs 23-25, wherein the cancer is        a leukemia, a lymphoma, multiple myeloma, or a solid tumor.    -   31. The method of paragraph 30, wherein the leukemia is acute        lymphocytic leukemia (ALL)    -   30 or chronic lymphocytic leukemia (CLL).    -   32. The method of paragraph 30, wherein the lymphoma is        follicular lymphoma or diffuse large B cell lymphoma (DLBCL).

EXAMPLES

Described herein is a novel technology to induce T cell proliferationand/or activation. These methods described in the following examplesutilize gamma-irradiated artificial antigen-presenting cells (aAPCs)genetically engineered to constitutively express chimeric stimulatoryreceptors (CSR) specifically recognizing both CD3 and CD28 (FIG. 1 ).The feasibility and efficacy of aAPC in which antibody constructsbinding both molecules are loaded onto the aAPC surface are demonstratedherein; the genetically engineered form described bypasses the need forclinical-grade soluble antibodies as accessory reagents.

Further contemplated herein is the use of the described technology forcommercial applications in cellular immunotherapy.

CD3/CD28 microspheres to expand T cells are very costly ($6,000 pervial), representing a significant expense in cell manufacturing.Moreover, the process of “bead removal” prior to aliquotting into unitdoses for cryopreservation and final use, represents an additionalonerous step in T cell manufacturing that poses high contamination risksand increases the production costs. Microspheres can also providecontinuous stimulation to T cells in culture which, accompanied by thepresence of high concentrations of exogenous IL-2, may induce T cellexhaustion, which would be counter-productive in anti-cancer therapies.

In contrast, the aAPCs described herein can be manufactured as aclinical (GMP) grade reagent, expanded from an initial master cell bankand cryopreserved in production batches following gamma-irradiation torender them non-dividing. More importantly, aAPC of this nature can bereadily used after thawing, do not proliferate in culture, and can becompletely eliminated if an additional target molecule is co-expressedon their surface (e.g., CD19 as target for CART19 cells). aAPCsdescribed in this example and elsewhere herein, are engineered toexpress CSRs, which bypasses the need to use clinical grade antibodies.There is also no need for additional steps to purify T-cell products atthe end of manufacturing runs, making these aAPC an attractive andcost-effective new technology in the production of engineered T cellsfor clinical therapies.

The methods described herein can be utilized to treat cancer patientswith leukemias such as ALL or CLL, with lymphoma (follicular or DLBCL),or with multiple myeloma, each of which have been successfully treatedwith CART cell therapies. It is further contemplated that the patientpopulation can be expanded to patients with solid tumors.

Example 1 Methods

Production of aAPC Co-Expressing CD3- and CD28-Targeting CSR (Chimeric35 Stimulatory Receptors).

CSR-CD3 (derived from an anti-CD3 Mab) and CSR-CD28 (from an anti-CD28MAb) sequences are cloned into lentiviral vector (pMGH vector). Cells tobe used as aAPC (K562, erythromyeloid cell line) are transduced withlentiviruses encoding pMGH-CSR-CD3-T2A-GFP andpMGH-CSR-CD28-T2A-mCherry. The fluorescent reporters will be used toFACS sort the aAPC that co-express both CSRs. Fluorescent reporters haveno effect on the stability of the aAPC system. aAPC are expanded,gamma-irradiated and cryopreserved (5-10e6 cells/vial). aAPC arevalidated (cell viability and cell numbers, and FACS analysis for theexpression of CSR-CD3 and CSR-CD28 post-thawing and at multiple timepoints during culture).

Analysis of Engineered T-Cell Products Manufactured with aAPC.

The efficiency of aAPC to stimulate primary T cells is determined, e.g.,by measuring T-cell growth with expansion curves as shown in FIG. 2 .Briefly, T cells are seeded at day 0 at a 3:1 ratio (beads:T cells) andat different aAPC:T cell ratios (optimal ratio will be determined).Total cell numbers and mean cell volumes are measured at days 3, 5, 7,and 9 using a MultisizerTM 3 Coulter Counter (Beckman Coulter), andcellular viability is determined by staining with AcridineOrange/Propidiumlodide exclusion dye using a Luna-FLTM Cell Counter(Logos Biosystems).

Analysis of transduction efficiency of primary T cells co-cultured withaAPC. FACS analysis is performed to determine transduction efficiency.For example, when using the CAR19 lentiviral vector (recognizing CD 19),streptavidin-conjugated protein L can be used to detect the single chainvariable fragment of the CAR.

Potency tests of T cell products manufactured in the presence of aAPC;comparison with products made with CD3/CD28 microspheres. Functionalcharacteristics are measured on transduced T cells upon re-stimulationwith target-antigen (eg. CD19, BCMA, etc.) expressing cells:

-   -   Proliferative capacity (CFSE dilution and absolute cell numbers        by FACS using fluorescently-labeled counting beads).    -   Cytokine production (10-Piex Luminex Assays).    -   Target-cell killing capacity (Bioluminesce analysis of target        cells in vitro or animal model system to track both tumor and        engineered T cells infused in immunodeficient mice).    -   Cell degranulation analysis (CD 107a release assay in response        to target cells, measured by FACS).

CSR Construction

The CD3 CSR (OKT3) comprised, in addition to an ScFv that binds CD3:

-   -   CD8 hinge and TM domain;    -   CD3 (5 first aminoacids);    -   T2A; and    -   GFP.

The CD28 CSR (9.3) comprised in addition to an ScFv that binds CD28:

-   -   CD28 hinge-TM-ICD;    -   T2A; and    -   mCherry.

Artificial Antigen Presenting Cells (aAPC) were designed to express twoCSR (transgene structure shown in FIG. 1 ): CD3 CSR (OKT3 in both scFvconfigurations heavy-light and light-heavy). Reporter gene =GFP. CD28CSR (clone 9.3 in light-heavy scFv configuration). Reporter gene=mCherry. Three lentiviruses were generated:

-   -   pMGH43=CD3 CSR (L-H)    -   pMGH44=CD3 CSR (H-L)    -   pMGH45−CD28 CSR (L-H)

aAPCs were single-cell sorted based on GFP and mCherry expression (FIG.4 ), expanded and cryopreserved. Jurkat cells (a human T-cell line),expressing a luciferase gene under the control of an NFAT promoter weretransduced with two different CAR-encoding lentiviruses. Jurkat cellswere co-cultured for 18 hours with wild-type K562 cells (negativecontrol), Dynabeads coated with anti-CD3 and anti-CD28 antibodies(positive control), and aAPCs (expressing both CD3 and CD28 CSRs). aAPCsinduced higher activation of the Jurkat cells than Dynabeads asdetermined by the higher relative luminescence units (RLU) (FIG. 5 ).Surprisingly, aAPCs (expressing CD3+CD28 CSRs) promote more humanprimary T-cell growth than the conventional method using Dynabeads (FIG.6 ; 1 donor and FIG. 7 ; 3 normal donors). Human T cells expand duringactivation with aAPCs during the first three days of culture, andsubsequently contract following a similar pattern as when T cells arestimulated with Dynabeads (FIG. 8 ).

Generation and Validation of LDLR KO aAPCs Using Electroporated K562Cells

Expression of the low-density lipoprotein receptor (LDLR) on cells iscritical for the attachment and entrance of VSV-g-pseudotyped viruses.Using CRISPR, we knocked out the expression of the LDLR on K562 cells tobe used as artificial antigen presenting cells (aAPC) to enhance thetransduction efficiency of genetically-modified cells using a lentiviralsystem. The KO was validated as follows:

-   -   Nucleases analyzed:        -   SpCas9        -   SpCas9-H1 (High fidelity)        -   AsCpfl        -   LbCpfl    -   RNA Guides analyzed:        -   9→SpCas9        -   4→Cpfl    -   Total number of samples/cultures=54+24=78        -   Cas9: 2 nucleases×9 guides=18→In triplicates: 18×3=54        -   Cpfl: 2 nucleases×4 guides=8→In triplicates: 8×3=24    -   Validation of indels in extracted genomic DNA of K562 cells:        -   T7E1 assay (denature DNA-Annealing-Gel            electrophoresis-Calculation        -   WT:digested DNA bands        -   FACS (LDLR-PE surface staining)

The CRISPR technology was used to disrupt the expression of the LDLR onaAPCs. Four DNA nucleases (wt Cas9, Cas9 HF1, AsCpfl, and LbCpfl) andten different RNA guides were tested. FIG. 9 , top panel shows theresults from a T7E1 assay indicating the percent disruption ormodification level achieved on the LDLR gene using a specificcombination of nuclease and guide RNA. The highest % disruptionefficiencies were obtained using the following combinations:

-   -   1. Cas9 (both wt and HF1)+Exon1-Site1 guide RNA    -   2. Cpfl (both As and Lb)+Exon1-Site1 guide RNA    -   3. Cpfl (both As and Lb)+Exon3-Site2 guide RNA

The results from the T7E1 assay were validated by flow cytometry,staining cells with an anti-human LDLR-PE antibody (FIG. 9 , bottompanel). FACS controls were cells electroporated with nuclease-encodingDNA without guide RNA.

FIG. 10 depicts FACS results for 10 selected clones after single-cellsorting and expansion (>20 days). Homogeneous populations were obtained.In some of these clones, the disruption efficiency achieved was veryhigh (eg. sample #9; 86.7%) 2{circumflex over ( )}(15 days)=32,000 cells

CRISPR targeting the LDLR gene was repeated for aAPCs exressing CD3+CD28 CSRs. High levels of gene disruption were achieved using thenuclease-guide RNA combinations from the previous assay (FIG. 11 ).Results of FACS analysis with anti-human LDLR-PE antibody are depictedin FIG. 12 for the nuclease +gRNA combinationof AsCpfl+LDLR Exon3, Site3 gRNA. The LDLR KO aAPCs were confirmed by flow cytometry to expressCD3 and CD8 CSRs (FIG. 13A) but not LDLR (FIG. 13B).

Both LDLR+and LDLR KO aAPCs were transduced with a GFP-encodinglentivirus. Transduction efficiency (measured by % GFP cells) issignificantly reduced in LDLR KO aAPCs (FIGS. 14A and 14B). Whenpurified T cells from normal donors were stimulated with either anti-CD3and anti-CD28 antibody-conjugated beads (bCD3/CD28, 3:1) or LDLR KOaAPCs (1:1) and analyzed by FACS five days post-stimulation, a highertransduction efficiency was observed using the LDLR KO aAPCs as comparedto the using bCD3/CD28 (FIGS. 15A and 15B). However, CAR T cellsexpanded with the aAPCs share similar cytokine profiles compared to CART cells expanded with bCD3/CD28 (FIG. 16 ). Additionally, CAR T cellsexpanded with the aAPCs exhibit similar tumor killing capacity comparedto CAR T cells expanded with bCD3/CD28, both in vitro (FIG. 17 ) and invivo (FIG. 18 ).

Example 2

aAPC can Induce Human T-Cell Proliferation

The capacity of cell-based aAPC to stimulate human T cells and inducetheir proliferation has been tested as follows. Primary T cells wereisolated from peripheral blood of a normal donor and a chroniclymphocytic leukemia patient. Both T cell preparations were cultured for9 days in the presence of CD3/CD28 microspheres (current method) or withaAPC displaying anti-CD3 and anti-CD28 antibody constructs. T-cellsco-cultured with aAPC expanded in a similar or even better fashion thanthe ones stimulated with the microspheres (FIG. 2 ). Similar resultswere obtained by measuring [³H]thymidine incorporation in bothpolyclonal CD4+ and CD8+ T cells purified from multiple donors activatedeither by aAPC or microspheres (FIG. 3 ).

Example 3

CSR construct sequencespMGH43-anti-hOKT3-L/H-GFP (SEQ ID NO: 1) comprisingCD8leader (amino acids 1-21 (SEQ ID NO: 2)); VL(amino acids 22-127 (SEQ ID NO: 3)); linker (aminoacids 128-147 (SEQ ID NO: 4)), VH (amino acids148-266 (SEQ ID NO: 5)), CD8hingeTM (amino acids267-335 (SEQ ID NO: 6)); AAAAA-CD3Z (amino acids336-340 (SEQ ID NO: 7)); T2A linker (amino acids341-364 (SEQ ID NO: 8)); and GFP (amino acids 365-603 (SEQ ID NO: 9)).(SEQ ID NO: 1)MALPVTALLLPLALLLHAARPDIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSKLASGVPSRFSGSGSGTDYTFTISSLOPEDIATYYCQQWSSNPFTFGQGTKLQITGGGGSGGGGSGGGGSGGGGSQVQLVQSGGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGKGLEWIGYINPSRGYTNYNQKVKDRFTISRDNSKNTAFLQMDSLRPEDTGVYFCARYYDDHYCLDYWGQGTPVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRVKFSSGGGGEGRGSLLTCGDVEENPGPRMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKCD8 leader (SEQ ID NO: 2 (amino acids 1-21 of SEQ ID NO: 1))(SEQ ID NO: 2) MALPVTALLLPLALLLHAARPVL sequence (SEQ ID NO: 3 (amino acids 22-127 of SEQ ID NO: 1))(SEQ ID NO: 3)DIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSKLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGQGTKLQITLinker sequence (SEQ ID NO: 4 (amino acids 128-147 of SEQ ID NO: 1))(SEQ ID NO: 4) GGGGSGGGGSGGGGSGGGGSVH sequence (SEQ ID NO: 5 (amino acids 148-266 of SEQ ID NO: 1))(SEQ ID NO: 5) KDRFTISRDNSKNTAFLQMDSLRPEDTGVYFCARYYDDHYCLDYWGQGTPVTVSSCD8 hingeTM (SEQ ID NO: 6 (amino acids 267-335 of SEQ ID NO: 1))(SEQ ID NO: 6)TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLS LVITLYCAAAAA-CD3Z (SEQ ID NO: 7 (amino acids 336-340 of SEQ ID NO: 1))(SEQ ID NO: 7) RVKFS T2A linker (SEQ ID NO: 8 (amino acids 341-364 ofSEQ ID NO: 1)) (SEQ ID NO: 8) SGGGGEGRGSLLTCGDVEENPGPRGFP (SEQ ID NO: 9 (amino acids 365-603 of SEQ ID NO: 1)) (SEQ ID NO: 9)MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKpMGH44/anti-hOKT3_H/L-GFP (SEQ ID NO: 10) comprisingCD8leader (amino acids 1-21 (SEQ ID NO: 11)); VH(amino acids 22-140 (SEQ ID NO: 12)); linker (aminoacids 141-160 (SEQ ID NO: 13)); VL (amino acids161-266 (SEQ ID NO: 14)); CD8hingeTM (amino acids267-335 (SEQ ID NO: 15)); AAAAA-CD3Z (amino acids336-340 (SEQ ID NO: 16)); T2A (amino acids 341-364(SEQ ID NO: 17)); and GFP (amino acids 365-603 (SEQ ID NO: 18)).(SEQ ID NO: 10)MALPVTALLLPLALLLHAARPQVQLVQSGGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGKGLEWIGYINPSRGYTNYNQKVKDRFTISRDNSKNTAFLQMDSLRPEDTGVYFCARYYDDHYCLDYWGQGTPVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSKLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGQGTKLQITTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRVKFSSGGGGEGRGSLLTCGDVEENPGPRMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKCD8 leader (SEQ ID NO: 11 (amino acids 1-21 of SEQ ID NO: 10))(SEQ ID NO: 11) MALPVTALLLPLALLLHAARPVH sequence (SEQ ID NO: 12 (amino acids 22-140 of SEQ ID NO: 10))(SEQ ID NO: 12) KDRFTISRDNSKNTAFLQMDSLRPEDTGVYFCARYYDDHYCLDYWGQGTPVTVSSLinker (SEQ ID NO: 13 (amino acids 141-160 of SEQ ID NO: 10))(SEQ ID NO: 13) GGGGSGGGGSGGGGSGGGGSVL sequence (SEQ ID NO: 14 (amino acids 161-266 of SEQ ID NO: 10))(SEQ ID NO: 14)DIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSKLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGQGTKLQITCD8 hingeTM (SEQ ID NO: 15 (amino acids 267-335 of SEQ ID NO: 10))(SEQ ID NO: 15)TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLS LVITLYCAAAAA-CD3Z (SEQ ID NO: 16 (amino acids 336-340 of SEQ ID NO: 10))(SEQ ID NO: 16) RVKFS T2A linker (SEQ ID NO: 17 (amino acids 341-364 ofSEQ ID NO: 10)) (SEQ ID NO: 17) SGGGGEGRGSLLTCGDVEENPGPRGFP (SEQ ID NO: 18 (amino acids 365-603 of SEQ ID NO: 10))(SEQ ID NO: 18)MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKpMGH45/anti-9.3-CD28-T2A-mCHERRY (SEQ ID NO: 19)comprising CD8leader (amino acids 1-20 (SEQ IDNO: 20)); scFv-CD28-VL (amino acids 21-133 (SEQID NO: 21)); linker (amino acids 134-153 (SEQ IDNO: 22)); scFv-CD28-VH (amino acids 154-275 (SEQID NO: 23)); CD28-EC-TM-ICD (amino acids 276-358(SEQ ID NO: 24)); T2A (amino acids 359-382 (SEQID NO: 25)); and mCHERRY (amino acids 383-618 (SEQ ID NO: 26)).(SEQ ID NO: 19)MESDTLLLWVLLLWVPGSTGDIVLTQSPASLAVSLGQRATISCRASESVEYYVTSLMQWYQQKPGQPPKLLIFAASNVESGVPARFSGSGSGTNFSLNIHPVDEDDVAMYFCQQSRKVPYTFGGGTKLEIKRAGGGGSGGGGSGGGGSGGGGSLAQVQLKESGPGLVTPSQSLSITCTVSGFSLSDYGVHWVRQSPGQGLECLGVIWAGGGTNYNSALMSRKSISKDNSKGQVFLKMKSLQADDTAVYYCARDKGYSYYYSMDYWGQGTSVTVSSKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSSGGGGEGRGSLLTCGDVEENPGPRMVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKCD8 leader (SEQ ID NO: 20 (amino acids 1-20 of SEQ ID NO: 19))(SEQ ID NO: 20) MALPVTALLLPLALLLHAARPscFv-CD28-VL (SEQ ID NO: 21 (amino acids 21-133 of SEQ ID NO: 19))(SEQ ID NO: 21)DIVLTQSPASLAVSLGQRATISCRASESVEYYVTSLMQWYQQKPGQPPKLLIFAASNVESGVPARFSGSGSGTNFSLNIHPVDEDDVAMYFCQQSRKVPYTFGGGTKLEIKRALinker (SEQ ID NO: 22 (amino acids 134-153 of SEQ ID NO: 19))(SEQ ID NO: 22) GGGGSGGGGSGGGGSGGGGSscFv-CD28-VH (SEQ ID NO: 23 (amino acids 154-275 of SEQ ID NO: 19))(SEQ ID NO: 23)LAQVOLKESGPGLVTPSQSLSITCTVSGFSLSDYGVHWVRQSPGQGLECLGVIWAGGGTNYNSALMSRKSISKDNSKGQVFLKMKSLQADDTAVYYCARDKGYSYYYSMDYWGQGTSVTVSSCD28-EC-TM-ICD (SEQ ID NO: 24 (amino acids 276-358 of SEQ ID NO: 19))(SEQ ID NO: 24)KHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS T2A linker (SEQ ID NO: 25 (amino acids 359-382 ofSEQ ID NO: 19)) (SEQ ID NO: 25) SGGGGEGRGSLLTCGDVEENPGPRmCherry (SEQ ID NO: 26 (amino acids 383-618 of SEQ ID NO: 19))(SEQ ID NO: 26)MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYK

Example 4

Antibody sequences OKT3 L/H (SEQ ID NO: 27) comprising VL (amino acids1-106 (SEQ ID NO: 28)); linker (amino acids 107-126(SEQ ID NO: 29)); and VH (amino acids 127-245 (SEQ ID NO: 30)).(SEQ ID NO: 27)DIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSKLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGQGTKLQITGGGGSGGGGSGGGGSGGGGSQVQLVQSGGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGKGLEWIGYINPSRGYTNYNQKVKDRFTISRDNSKNTAFLQMDSLRPEDTGVYFCARYYDDHYCLDYWGQGTPVTVSSVL (SEQ ID NO: 28 (amino acids 1-106 of SEQ ID NO: 27)) (SEQ ID NO: 28)DIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSKLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGQGTKLQITLinker (SEQ ID NO: 29 (amino acids 107-126 of SEQ ID NO: 27))(SEQ ID NO: 29) GGGGSGGGGSGGGGSGGGGSVH (SEQ ID NO: 30 (amino acids 127-245 of SEQ ID NO: 27))(SEQ ID NO: 30)QVQLVQSGGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGKGLEWIGYINPSRGYTNYNQKVKDRFTISRDNSKNTAFLQMDSLRPEDTGVYFCARYYDDHYCLDYWGQGTPVTVSSOKT3 H/L (SEQ ID NO: 31) comprising VH (amino acids1-119 (SEQ ID NO: 32)); linker (amino acids 120-139(SEQ ID NO: 33)); and VL (amino acids 140-245 (SEQ ID NO: 34)).(SEQ ID NO: 31)QVQLVQSGGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGKGLEWIGYINPSRGYTNYNQKVKDRFTISRDNSKNTAFLQMDSLRPEDTGVYFCARYYDDHYCLDYWGQGTPVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSKLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGQGTKLQITVH (SEQ ID NO: 32 (amino acids 1-119 of SEQ ID NO: 31)) (SEQ ID NO: 32)KVKDRFTISRDNSKNTAFLQMDSLRPEDTGVYFCARYYDDHYCLDYWGQGTPVTVSSLinker (SEQ ID NO: 33 (amino acids 120-139 of SEQ ID NO: 31))(SEQ ID NO: 33) GGGGSGGGGSGGGGSGGGGSVL (SEQ ID NO: 34 (amino acids 140-245 of SEQ ID NO: 31))(SEQ ID NO: 34)DIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSKLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGQGTKLQITanti-CD28 (9.3 L/H) (SEQ ID NO: 35) comprising VL(amino acids 1-113 (SEQ ID NO: 36)); linker (aminoacids 114-133 (SEQ ID NO: 37)); and VH (amino acids134-255 (SEQ ID NO: 38)). (SEQ ID NO: 35)DIVLTQSPASLAVSLGQRATISCRASESVEYYVTSLMQWYQQKPGQPPKLLIFAASNVESGVPARFSGSGSGTNFSLNIHPVDEDDVAMYFCQQSRKVPYTFGGGTKLEIKRAGGGGSGGGGSGGGGSGGGGSLAQVOLKESGPGLVTPSQSLSITCTVSGFSLSDYGVHWVRQSPGQGLECLGVIWAGGGTNYNSALMSRKSISKDNSKGQVFLKMKSLQADDTAVYYCARDKGYSYYYSMDYWGQG TSVTVSSVL (SEQ ID NO: 36 (amino acids 1-113 (SEQ ID NO: 35)) (SEQ ID NO: 36)DIVLTQSPASLAVSLGQRATISCRASESVEYYVTSLMQWYQQKPGQPPKLLIFAASNVESGVPARFSGSGSGTNFSLNIHPVDEDDVAMYFCQQSRKVPYTFGGGTKLEIKRALinker (SEQ ID NO: 37 (amino acids 114-133  (SEQ ID NO: 35))(SEQ ID NO: 37) GGGGSGGGGSGGGGSGGGGSVH (SEQ ID NO: 38 (amino acids 134-255 (SEQ ID NO: 35)) (SEQ ID NO: 38)LAQVQLKESGPGLVTPSQSLSITCTVSGFSLSDYGVHWVRQSPGQGLECLGVIWAGGGTNYNSALMSRKSISKDNSKGQVFLKMKSLQADDTAVYYCARDKGYSYYYSMDYWGQGTSVTVSS

1.-22. (canceled)
 23. A method of treating a cancer, a plasma celldisease or disorder, or an autoimmune disease or disorder in a subjectin need thereof, the method comprising: contacting an artificial antigenpresenting cell (aAPC) with a CAR T cell, thereby activating the CAR Tcell, wherein the aAPC comprises: a first chimeric stimulatory receptor(CSR) that binds specifically with a first co-stimulatory polypeptide;and a second CSR that binds specifically a second co-stimulatorypolypeptide; and administering the activated CAR T cell to the subject.24. The method of claim 23, wherein the contacting step occurs in vitro.25. The method of claim 23, wherein the contacting step occurs insuspension. 26.-29. (canceled)
 30. The method of claim 23, wherein thecancer is a leukemia, a lymphoma, multiple myeloma, or a solid tumor.31. The method of claim 30, wherein the leukemia is acute lymphocyticleukemia (ALL) or chronic lymphocytic leukemia (CLL).
 32. The method ofclaim 30, wherein the lymphoma is follicular lymphoma or diffuse large Bcell lymphoma (DLBCL).